Clathrin-Dependent Targeting of Receptors to the Flagellar Pocket of Procyclic-Form Trypanosoma brucei

Trypanosomes.The procyclic form of T. brucei was maintained at 25°C in SDM-79 medium supplemented with 10% fetal bovine serum (8). Bloodstream form trypanosomes were maintained in HMI-9 medium at 37°C (14). The procyclic trypanosome cell line 29-13 and the bloodstream form cell line 13-90, which harbor the T7 RNA polymerase gene and the tetracycline repressor gene, were obtained from G. Cross's laboratory and were used as the parental strains for transformation using tetracycline-inducible double-stranded RNA (dsRNA) expression constructs (49). For all transformations, early- to mid-log-phase trypanosomes (procyclic trypanosomes at a cell density of 5 × 10⁶ to 8 × 10⁶ cells/ml and the bloodstream form at a cell density of 2 × 10⁶ cells/ml) were used. For induction of dsRNA, trypanosomes were cultured in medium containing 1.0 μg of tetracycline per ml.

Antibodies.Polyclonal antibody against ESAG6 was a gift from the laboratories of Peter Overath and Piet Borst (27, 39). Monoclonal antibody p67 and the rabbit-derived polyclonal anti-Bip antibody were gifts from Jay Bangs (3, 15). Anti-CRAM antibody was as previously described (19). The rabbit-derived anti-TbCLH polyclonal antibody was raised against a glutathione S-transferase-TbCLH recombinant protein containing the TbCLH coding region spanning from amino acids 761 to 1007. The monoclonal anti-EP procyclin (containing glutamic acid [E]-proline [P] repeats) antibody was purchased from Cedarlane. The mouse-derived anti-α-tubulin monoclonal antibody was purchased from Sigma Co. The Golgi-marker BODIPY TR ceramide was purchased from Molecular Probes, Inc.

Stable DNA transformation.Linearized plasmid (10 or 20 μg) was electroporated into trypanosomes using a BTX electroporator as previously described (36). At 36 and 16 h after electroporation of the procyclic form and the bloodstream form, respectively, G418 (20 μg/ml for procyclics and 2 μg/ml for the bloodstream form), hygromycin B (40 μg/ml for procyclics and 2 μg/ml for the bloodstream form), and phleomycin (2 μg/ml for procyclics and 1 μg/ml for the bloodstream form) were added to select stably transformed trypanosomes. The individually cloned drug-resistant trypanosomes were obtained by limiting dilution using microtiter dishes. To clone procyclic transformants, 2 × 10⁶ to 5 × 10⁶ wild-type trypanosomes per ml were added to each well to facilitate cell growth.

RNA isolation and Northern blot analysis.All RNA samples were isolated by guanidine thiocyanate lysis and purified by centrifugation through CsCl cushions. RNA samples were separated in 1% formaldehyde-agarose gels and transferred to nitrocellulose filters. Northern blots were hybridized with ³²p-labeled probes. Following hybridization, filters were washed to a final stringency of 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate at 65°C.

Western blot analysis and ELISA.Total-cell lysates of 2 × 10⁷ trypanosomes were size separated in polyacrylamide gels and electrophoretically transferred to nitrocellulose filters. The nitrocellulose filters were blocked with 5% nonfat milk in TBST (50 mM Tris-buffered saline, 0.05 or 0.1% Tween) and then reacted with the first antibodies in TBST with 5% nonfat milk for 1 h at room temperature. After three washes with TBST, the filters were treated with the horseradish peroxidase-labeled secondary antibody for 1 h. After three washes, the filter was reacted with an enhanced chemiluminescence detection system (Amersham Life Science). The protein level was quantitated by enzyme-linked immunosorbent assay (ELISA) analysis. Each well of 96-well ELISA plates was coated with 100 μl of total protein lysates of 1.5 × 10⁵ trypanosomes, incubated overnight at 4°C, and then blocked with 3% nonfat milk in phosphate-buffered saline (PBS)-0.1% Tween 20 (PBST) for 1 h at room temperature. Following three washes, the plates were incubated for 1.5 h at room temperature with primary antibodies (100 μl/well) which were diluted in PBST-3% nonfat milk. After the wells were washed, 100 μl of horseradish peroxidase-conjugated secondary antibody (diluted 1:10⁴ in PBST-3% nonfat milk) was added to each well, and the wells were incubated for 1.5 h at room temperature and then washed. To develop the plates, 100 μl of 3,3′,5,5′- tetramethylbenzidine (TMB) solution (Sigma Co.) was added to each well. The reaction was stopped by the addition of 50 μl of 2 M H₂SO₄ per well, and the optical density was measured at 450 nm with an ELISA photometer (MR5000; Dynatech).

Immunofluorescence microscopy.Trypanosomes were harvested by centrifugation for 10 min at 400 × g and washed once with PBS. The cells were suspended in 4% paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature. Following fixation, the cells were dotted onto 12- well slides and fixed in cold methanol for 5 min. After rehydration in glycine (0.14 M in PBS), the slides were blocked with 3% bovine serum albumin (BSA) in PBS for 1 h. Then the first antibody reactions were performed with PBS-0.1% Tween 20-3% BSA for 1 h and then slides were washed three times with PBS. The slides were then reacted with various fluorophore-conjugated goat-derived anti-rabbit, anti-rat, or anti-mouse immunoglobulin G (IgG) for 1 h. After being washed with PBS, the slides were mounted with mounting medium containing 4′,6- diamino-2-phenylindole (DAPI) (H-1200; Vector Laboratories Inc.). The cells were viewed and photographed under a Nikon or Leica fluorescence microscope. The images were directly captured by a charge-coupled device (CCD) camera and analyzed by the MetaMorph program of Universal Imaging Co.

Serum lipoprotein purification.Different lipoprotein fractions from bovine serum were isolated by sequential flotation ultracentrifugation (13). The range of densities recovered for high-density lipoprotein particles (HDL) is 1.063 to 1.21. Protein concentrations were determined using the bicinchoninic acid protein assay reagent (Pierce). Lipoproteins were ¹²⁵I labeled using IODO-GEN iodination reagent and precoated tubes (Pierce).

Purification and labeling of IgG.IgG molecules from immunized rabbit serum were purified by using the Affi-Gel protein A maps II kit (Bio-Rad). The purified IgGs were concentrated using Centricon YM-30 (Millipore). IgG was labeled with ¹²⁵I by using IODO-GEN iodination reagent (Pierce). Nonimmune rabbit IgG was purchased from Sigma Co.

Uptake and degradation.For each assay, 1 or 0.5 ml of cells (10⁷ cells/ml) were used. For the uptake assay; ¹²⁵I-labeled ligand (IgG or HDL) was incubated at 28°C for 2 to 4 h with trypanosomes in SDM-79 serum-free medium-3% BSA in the absence or presence of a 20-fold excess of unlabeled ligands. Following incubation, the trypanosomes were washed three times with SDM-79 medium-0.2% BSA. They were then transferred to a new set of tubes and centrifuged, and the amount of radioactivity associated with the cell pellets was measured in a gamma counter and referred to as the amount of uptake.

For the degradation assay, following the incubation of trypanosomes with ¹²⁵I-ligand at 28°C, trypanosomes were centrifuged and the supernatants were collected for determination of the amount of trichloroacetic acid (TCA; 10%, wt/vol)-soluble ¹²⁵I-labeled products. The amount of free ¹²⁵I was removed from the TCA-soluble fraction by precipitation with 5% (wt/vol) silver nitrate. The amount of radioactivity in the TCA-soluble, noniodide fraction was referred to as the amount of degradation. Total and nonspecific counts were referred to as the measurement of reactions performed in the absence and presence, respectively, of a 20-fold excess of unlabeled ligands. Specific counts were obtained by subtraction of nonspecific counts from total counts.

TbCLH RNAi reduces the growth efficiency of trypanosomes.We used the dsRNA interference (RNAi) approach to evaluate the function of TbCLH in T. brucei. The original TbCLH probe was obtained from the T. brucei genome project. Following the screening of a cDNA library and reverse transcription-PCR, we obtained the full-length TbCLH cDNA clone. We used the pZJM vector, provided by P. Englund's laboratory, to express dsRNA of TbCLH in trypanosomes (43). A DNA fragment of 738 bp, encoding amino acids 761 to 1007 of TbCLH, was cloned into pZJM vector, resulting in pZJM- CLH, in which the TbCLH dsRNA expression is controlled by two opposing and inducible T7 promoter-tetracycline operators (43). The plasmid is designed to integrate into the rDNA nontranscribed spacer region. The linearized pZJM-CLH was introduced into the bloodstream form cell line 13-90 and the procyclic-form cell line 29-13 which express T7 RNA polymerase and tetracycline repressor (49). Clonal transformed cell lines were established and maintained under noninducing conditions. Integration of the input plasmid at the nontranscribed rDNA spacer region in each cell line was confirmed by Southern blot analysis (data not shown). Then the impact of TbCLH RNAi on cell growth was studied after induction by the addition of tetracycline. We found that in bloodstream form trypanosomes, induction of TbCLH RNAi rapidly reduced cell growth; at 8 to 12 h after induction, most of the cells had rounded up and stopped replicating (Fig. 1A). In the procyclic form, at 24 h after induction of TbCLH RNAi, cell growth rapidly declined, although 97% of the cells remained morphologically unchanged (Fig. 1B; Table 1). At 48 h after TbCLH RNAi induction, procyclics started to die and a small portion of them (∼18%) adopted a round shape. At 72 h after induction of TbCLH RNAi, the majority of cells (∼90%) revealed a round morphology (Table 1). A representative image of the morphological changes of procyclics from the polarized shape to a nonpolar round shape is shown in Fig. 2, in which cells were stained with anti-α-tubulin antibody 70 h after induction of TbCLH RNAi. In addition to the cell shape change, these round cells appeared to lack the long extended flagellum (Fig. 2). Currently, we are not certain about the mechanisms involved in the shortening of the flagellum. However, we speculated that TbCLH RNAi might have impaired the biogenesis of the flagellar pocket and the flagellum. To confirm the RNAi effect, we compared the steady-state TbCLH mRNA levels in TbCLH RNAi-induced and -noninduced trypanosomes by Northern blot analysis (Fig. 1, bottom panels). Induction of TbCLH RNAi for 12 h in the bloodstream form and for 24 h in the procyclic form indeed drastically reduced the steady-state mRNA level of TbCLH, while the αβ-tubulin mRNA level was not significantly changed (Fig. 1, bottom panels).

• Open in new tab
• Download powerpoint

FIG. 1.

Effect of TbCLH RNAi on cell growth of T. brucei. A cell population derived from an individually transformed drug-resistant trypanosome was used for the growth analysis. The growth curves for the TbCLH RNAi trypanosome cell lines (the bloodstream form [A] and the procyclic form [B]) were determined in the absence (black squares; tet−) or presence (white squares; tet+) of 1 μg of tetracycline per ml (for induction of dsRNA expression). Cells were continuously maintained at log phase. If needed, medium was added to expand the culture. The total number of cells in each culture was calculated at different time points. The bottom panels represent the Northern blot analysis of the effects of RNAi. TbCLH and Tubulin indicate the probes used. 13-90 indicates that the RNA sample was obtained from the parental bloodstream cell line 13-90. − and + indicate that RNA samples were obtained from cells cultured in the absence or presence of tetracycline for 12 h (for the bloodstream form) or 24 h (for the procyclic TbCLH RNAi cell line), respectively.

• Open in new tab
• Download powerpoint

FIG. 2.

Morphological changes of procyclic trypanosomes on induction of TbCLH RNAi. Trypanosomes were first incubated with a monoclonal anti-α- tubulin antibody and then reacted with rhodamine-conjugated goat anti-mouse IgG. Slides were mounted with mounting medium containing DAPI. The large and small blue dots in each cell represent the nucleus and the kinetoplast, respectively. (A) Procyclic trypanosome with a wild-type shape. (B) Round cells that developed 70 h after the induction of TbCLH RNAi.

The rapid and severe lethality of TbCLH RNAi to bloodstream form trypanosomes has hampered our planned biochemical analysis of the bloodstream form TbCLH RNAi cell lines. The impact of TbCLH RNAi on the morphological changes of the bloodstream form trypanosomes was previously described in detail by Allen et al. (1). Therefore, we will not reiterate these observations in this report. However, we were surprised by the rapid and dramatic killing effect by TbCLH RNAi in procyclics, in which TbCLH expression is relatively low and the formation of large coated vesicles may not be a prerequisite for endocytosis. We speculated that TbCLH may have unique and essential functions in procyclics and subsequently focused our studies on the function of TbCLH in protein trafficking in procyclic-form trypanosomes.

TbCLH RNAi reduces the endocytosis efficiency of the procyclic form of T. brucei.We first determined the impact of TbCLH RNAi in endocytosis and protein trafficking in procyclics, for which very few studies of the related issues have been documented. We have previously demonstrated that the procyclic form of T. brucei takes up lipoprotein particles via receptor-mediated endocytosis (20) and characterized CRAM receptor-mediated endocytosis in procyclics by using anti-CRAM IgG as a ligand (22). Therefore, we chose to compare the endocytic efficiency of HDL particles and anti-CRAM IgG in procyclics before and after 24 h of TbCLH RNAi induction (at this point, most of the procyclics still have the wild-type morphology). It appeared that induction of TbCLH RNAi for 24 h specifically reduced the uptake of ¹²⁵I-anti-CRAM to 40% (Fig. 3A) and that of ¹²⁵I- HDL to 35% (Fig. 3C) in TbCLH RNAi procyclic cell lines compared to the uptake observed in noninduced cells. As a control, the uptake ability of the parental host cell line 29-13 in the presence and absence of tetracycline was measured (Fig. 3, bars 1− and 1+). Addition of tetracycline alone did not significantly affect the uptake efficiency of the cell line 29-13 (Fig. 3, bars 1+). We next determined whether the cell-associated ligands can be efficiently internalized and degraded upon induction of TbCLH RNAi. Following incubation of trypanosomes with ¹²⁵I- anti-CRAM and ¹²⁵I-HDL, the supernatant was harvested and the amount of TCA-soluble noniodide material that represents degradation products was measured. Interestingly, degraded materials could not be detected after induction of TbCLH RNAi for 24 h (Fig. 3B B and D). The control experiments using cell line 29-13 showed that addition of tetracycline alone did not significantly affect the degradation efficiency of procyclics (Fig. 3B and D and bars 1+). Thus, down-regulation of TbCLH drastically reduced the uptake of macromolecules and completely abolished the degradation ability of procyclics. Even though trypanosomes retained 35 to 40% of their ability to bind ligand molecules, on induction of TbCLH RNAi, these trypanosomes were incapable of internalizing and/or directing the bound ligands to endosomal/lysosomal compartments. These results suggested that TbCLH plays important roles in receptor-mediated endocytosis in procyclic trypanosomes.

• Open in new tab
• Download powerpoint

FIG. 3.

Reduction of the efficiency of receptor-mediated endocytosis in procyclic-form trypanosomes by TbCLH RNAi. Procyclic TbCLH RNAi trypanosomes (10⁷ cells/ml) were incubated at 28°C for 2 h with 60 μg of ¹²⁵I-anti-CRAM IgG per ml (A and B) or with 100 μg of ¹²⁵I-HDL per ml (C and D) in serum-free SDM-79 medium containing 3% (wt/vol) BSA. The cell-associated counts were then measured and referred to as the amount of uptake (A and C). The supernatants were collected for the measurement of degradation products (B and D). The results represent the amount of specific uptake and degradation, calculated as the difference between total (in the absence of unlabeled anti-CRAM IgG or HDL) and nonspecific uptake and degradation, respectively (in the presence of 20-fold unlabeled anti-CRAM IgG or 1 mg of unlabeled HDL per ml, respectively). Results represent the average of triplicate determinations. Bars 1 represent data generated by the host cell line 29-13, and bars 2 represent data generated by the procyclic TbCLH RNAi cell line. − and + indicate experiments performed before and after the addition of tetracycline for 24 h, respectively.

TbCLH RNAi affects the subcellular localization of CRAM in procyclic-form T. brucei.Our data have demonstrated that TbCLH is essential for cell growth and receptor- mediated endocytosis in procyclic trypanosomes, demonstrating its conserved role. We further evaluated the impact of TbCLH RNAi in the trafficking fate of receptor molecules in procyclics. Since currently CRAM is the only characterized receptor molecule at the flagellar pocket of procyclic trypanosomes, we compared the subcellular localization of CRAM before and after TbCLH RNAi induction by indirect-immunofluoresence analysis. It appeared that 24 h after the induction of TbCLH RNAi, ∼80% of the cells contained CRAM at the flagellar pocket while ∼20% exhibited aberrant localization of CRAM, which spread throughout the cell except for nucleus and kinetoplast (Fig. 4). The uptake analysis described above was performed at this time. Thus, a small amount of the reduction of uptake after TbCLH RNAi induction could be attributed to the lack of receptors at the flagellar pocket in some cells.

• Open in new tab
• Download powerpoint

FIG. 4.

TbCLH RNAi affecting the subcellular localization of CRAM in the procyclic form of T. brucei. Trypanosomes were incubated with the rabbit-derived anti-CRAM (19) antibody, reacted with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG, and then mounted with mounting medium containing DAPI. The images were captured using a CCD camera. The images are presented in pseudocolors: green for CRAM and blue for DAPI staining, which identifies the nucleus and the kinetoplast. The times 0, 24, 48, and 72 h indicate that the images were taken from cells prior to the induction of TbCLH RNAi and 24, 48, and 72 h after TbCLH RNAi induction, respectively.

Surprisingly, at 48 h after the induction of TbCLH RNAi, only 25% of cells exhibited flagellar pocket localization of CRAM; in 75% of cells, CRAM was no longer concentrated at the flagellar pocket but was spread throughout the cell (Fig. 4). At this point, morphologically, >80% of cells still maintained a flagellated, slender wild-type shape (Table 1). At 72 h after the induction of TbCLH RNAi, >95% of cells exhibited altered CRAM distribution and morphological changes to the round shape (Fig. 4). To determine whether TbCLH RNAi may result in the release of CRAM to the entire cell surface, we subsequently performed staining with anti-CRAM antibody on live trypanosomes at 4°C. We found that induction of TbCLH RNAi for 48 or 72 h did not lead to the escape of CRAM onto the cell surface or the surface of the flagellum (data not shown).

The aberrant CRAM distribution resulting from TbCLH RNAi frequently exhibits a reticulum or tubule structure and a distinct perinuclear staining pattern. This pattern is similar to the tubular network of the ER extending throughout the cell. To better understanding the altered CRAM distribution on TbCLH RNAi induction, we performed colocalization of CRAM with the ER-specific marker Bip, with the Golgi-specific dye BODIPY TR ceramide, and with the lysosomal/endosomal marker p67 (Fig. 5). We compared the colocalization of different markers in cells that retained their wild-type morphology after induction of TbCLH RNAi. Representative images are shown in Fig. 5. It was obvious that the majority of CRAM was colocalized with Bip at the ER after induction of TbCLH RNAi for 48 h. The colocalization of CRAM with BODIPY TR ceramide in the Golgi was reproducibly observed in some (∼50%) but not all cells after 48 h of induction of TbCLH RNAi. Figure 5B shows an example of cells that have no obvious localization of CRAM in the Golgi. These results indicated that down-regulation of TbCLH may have significantly reduced the efficiency of export of CRAM from the ER. It is possible that TbCLH may be required for targeting of many receptors or flagellar pocket proteins to the flagellar pocket in procyclics. However, this hypothesis cannot be tested until receptors other than CRAM are identified in procyclic trypanosomes.

• Open in new tab
• Download powerpoint

• Open in new tab
• Download powerpoint

FIG. 5.

Colocalization of CRAM with different markers in procyclics before and after TbCLH RNAi induction. (A) Colocalization analysis of procyclic cells prior to TbCLH RNAi induction. (B) Colocalization analysis of procyclics 48 h after TbCLH RNAi induction. Staining was performed using slides containing fixed and permeabilized trypanosomes. The slides were first incubated with different sets of primary antibodies and then reacted with various fluorophor-labeled secondary antibodies. After the slides were washed, cells were mounted with mounting medium containing DAPI. The images were captured using a CCD camera and analyzed by the MetaMorph program from Universal Imaging Co. The images are presented in pseudocolors: green for FITC-labeled CRAM, red for other rhodamine-labeled markers, and blue for DAPI staining, which identifies the nucleus and the kinetoplast. Panels p67 are images from cells costained with rabbit-derived anti-CRAM (green) and mouse monoclonal anti-p67 (red) antibodies. Panels Golgi are images from cells costained with anti-CRAM (green) and the Golgi- specific dye BODIPY TR ceramide (red). Panels Bip are images from cells stained with rat- derived anti-CRAM (green) and rabbit-derived anti-Bip (red) antibodies. Panels EP are images from cells stained with anti-CRAM (green) and the monoclonal anti-EP procyclin (red) antibody. Panels Tubulin are images from cells stained with anti-CRAM (green) and monoclonal anti-α- tubulin (red) antibodies.

We also determined whether TbCLH RNAi affects the transport of membrane proteins from the ER to the endosomal/lysosomal compartment. The distribution of p67 was compared in procyclics with and without TbCLH RNAi induction (Fig. 5). Overall, p67 remained located at a single specific area between the flagellar pocket and the nucleus on induction of TbCLH RNAi (Fig. 5). This result suggested that the trafficking of p67 most probably is not dependent on TbCLH. Additionally, we did not observe a significant amount of CRAM accumulating in the lysosomal/endosomal compartment.

Since surface coat proteins also travel from the ER to the Golgi and TGN and then to the flagellar pocket membrane and spread over the entire cell surface, as a comparison we compared the distribution of procyclin before and after the induction of TbCLH RNAi (Fig. 5). The procyclic 29-13-derived cell lines predominantly express the EP-procyclin. It appeared that induction of TbCLH RNAi did not significantly affect the distribution of the EP-procyclin over the entire cell surface, even though it prevented the export of CRAM from the ER to the flagellar pocket. This result suggested that at least two secretory pathways may exist in procyclic-form trypanosomes and that they can be distinguished by the requirement of TbCLH.

At 72 h after TbCLH RNAi induction, most of the procyclics had assumed an apolar round shape. The morphological changes have made it difficult to perform a precise evaluation of the colocalization. However, we found that p67 exhibited a highly dispersed distribution in round cells (data not shown). We speculate that at this stage, many morphological or physiological changes of various organelles along the trafficking pathway may directly or indirectly result from TbCLH RNAi.

TbCLH RNAi up-regulates the steady-state CRAM protein level in the procyclic form of T. brucei.Our studies suggested that TbCLH RNAi affected both inbound and outbound trafficking of CRAM in procyclics. These events may ultimately lead to a change of the turnover rate and the steady-state expression level of CRAM. In the subcellular localization experiments, we often observed that on induction of TbCLH RNAi, most cells exhibited a relatively intense staining with anti-CRAM antibody in addition to having an aberrant localization of CRAM. This observation suggests that either CRAM is more accessible to the antibody or the level of the CRAM protein is elevated. We thus compared the mobility and the steady- state expression level of CRAM before and after TbCLH RNAi induction by Western blot analysis. At 24, 48, and 72 h after induction of TbCLH RNAi, total-cell extracts from the same number of trypanosomes were analyzed (Fig. 6A). Surprisingly, we found that the level of CRAM protein expression was drastically increased on induction of TbCLH RNAi while the mobility of CRAM remained unchanged (Fig. 6A). These results indicated that down-regulation of TbCLH up-regulated the CRAM protein level and most probably did not significantly affect the posttranslational modifications of CRAM. In contrast, the protein level of procyclin and α-tubulin is not affected by TbCLH RNAi (Fig. 6A, bottom panels). We further quantitated the CRAM protein level by ELISA, using the level of α-tubulin protein as an internal control (Fig. 6B). We estimated that 24, 48, and 72 h of TbCLH RNAi induction increased the CRAM protein level by 1.3-, 2.5-, and 4.2-fold, respectively (Fig. 6B). To confirm the TbCLH RNAi effect, we measured the TbCLH protein level. At 24 h after induction, the TbCLH protein level was reduced to ∼30%, and at 48 and 72 h of induction, it had been reduced to effectively 0% (Fig. 6C).

• Open in new tab
• Download powerpoint

FIG. 6.

TbCLH RNAi up-regulates the steady-state CRAM protein level in the procyclic form of T. brucei. (A) Western blot analysis. Total-protein lysates derived from procyclic trypanosomes (∼2 × 10⁷ trypanosomes each) before (0) and 24, 48, and 72 h after TbCLH RNAi induction (indicated by 24 h, 48 h, and 72 h, respectively) were size separated in 6% polyacrylamide gels and electrophoretically transferred to nitrocellulose filters. The blots were probed with anti-CRAM antibody (panel CRAM). The same blots were later probed with anti- EP procyclin (panel EP) and anti-α-tubulin (panel Tubulin). Following the first antibody reaction, filters were treated with horseradish peroxidase-labeled goat anti-rabbit or anti- mouse IgGs. The signals were detected using an enhanced chemoluminescence detection system. (B) Quantitative analysis of the CRAM protein level by ELISA. ELISA was performed as described in Materials and Method. The left panel (CRAM) represents the amount of CRAM protein. The right panel (Tubulin) represents the amount of α-tubulin. Black bar, before induction; striped, grey, and white bars, 24, 48, and 72 h, respectively, after TbCLH RNAi induction. (C) Western blot analysis of TbCLH expression on induction of TbCLH RNAi. Total-protein lysates derived from procyclic trypanosomes (∼2 × 10⁷ trypanosomes each) before (0) and 24, 48, and 72 h after TbCLH RNAi induction (indicated by 24 h, 48 h, and 72 h, respectively) were size separated in 6% polyacrylamide gels and electrophoretically transferred to nitrocellulose filters. The blots were probed with anti-TbCLH antibody (panel TbCLH). The same blots were later probed with anti-α-tubulin (panel Tubulin) as a control to demonstrate the relative amount of protein loaded in each lane. −, extract from noninduced cells.