FIG. 3.

Immunological characterization of CSBP. (A) Specificity of antibodies against CSBPA and CSBPB. Immunoblots of Crithidia whole-cell extracts were probed with affinity-purified antibodies against CSBPA (lane 1) or CSBPB (lane 2). (B) Supershift of gel-retarded complexes by antibodies against CSBPA and CSBPB. Crithidia whole-cell extracts were preincubated for 15 min in the absence (lane 2) or presence of preimmune serum (lane 3), 0.5 μg of affinity-purified anti-CSBPA (lane 4), or 0.4 μg of affinity-purified anti-CSBPB (lane 5) or anti-SSE1 (lane 6) and anti-RPA1 (lane 7) antisera. Each reaction mixture contained approximately 0.5 μg of immunoglobulin G. The reaction mixtures were then further incubated for another 15 min at 30°C following the addition of ³²P-labeled 6X octameric RNA probe and analyzed by electrophoresis on a 4% nondenaturing polyacrylamide gel. Lane 1 shows probe alone in absence of cell extract. (C) Immunoprecipitation of CSBPA and CSBPB. Crithidia nuclear extract (300 μg) was used in immunoprecipitation reactions either with rabbit preimmune serum (lanes 1 and 6) or with 2.5 (lanes 2 and 7), 10 (lanes 3 and 8), or 40 (lanes 4 and 9) μg of affinity-purified polyclonal anti-CSBPA antibody. Immunoprecipitation reactions were also performed in the presence of 20 μg of RNase A (lanes 5 and 10) by using 40 μg of the anti-CSBPA antibody. The immunoprecipitates were then Western blotted and probed with either horseradish peroxidase-conjugated anti-CSBPB peptide antibody (lanes 1 to 5) or with biotinylated anti-CSBPA peptide antibody (lanes 6 to 10). The biotinylated antibody was detected with streptavidin-peroxidase.