Figure 3.

Ribosomal association of Pdcd4. (A) Cytoplasmic extracts isolated from HeLa cells were analyzed by sucrose density gradient centrifugation. Individual fractions of the gradient were then analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using antibodies against Pdcd4 (top) and eIF4A (middle) or by agarose gel electrophoresis and staining with ethidium bromide to detect 18S and 28S ribosomal RNAs (bottom). Fractions containing predominantly small ribosomal subunits, complete ribosomes, and polysomes are marked. (B) Cytoplasmic extracts from HEK293 cells were analyzed as described in panel A. (C) HeLa cells were analyzed as described in panel A except that EDTA was used to disrupt ribosomal complexes. (D) HeLa cells were analyzed as described in panel A except that the cell extract was treated with RNAse before centrifugation. (E) Cytoplasmic extracts of HeLa cells, pretreated for 30 min with or without cycloheximide, were immunoprecipitated with Pdcd4-specific antiserum or with preimmune serum. Coprecipitated 18S and 28S ribosomal RNAs were then visualized by agarose gel electrophoresis and staining with ethidium bromide. Aliquots of the samples were treated with DNase or RNase before gel electrophoresis. The bottom panel shows aliquots of the cytoplasmic extracts before immunoprecipitation.