FIG. 6.

p21 complexes with the 19S subunit together with MDMX or MDM2 in cells. (A and B) p21 interacts with S2 in cells. H1299 cells were transfected as indicated. Two micrograms of each plasmid was used. The transfected cells were treated with 10 μΜ MG132 for 16 h and harvested at 48 h posttransfection. The cell lysates (300 μg/sample) were immunoprecipitated with polyclonal anti-V5 antibody (A) or monoclonal anti-Flag antibody (B). The cell lysates (50 μg/sample) were also used for WB, as indicated on the right. (C) Molecular mass distribution profiles of overexpressed p21, S2, and MDMX or MDM2 after size exclusion chromatography. H1299 cells were transfected as indicated and treated with 10 μΜ MG132 for 16 h. The size exclusion chromatography was carried out as in Fig. 5A. The selected fractions (Frac; 30 μl/fraction) were revolved by 12% SDS-PAGE followed by WB, as indicated on the right. (D) p21 preferentially associates with S2 in the high-molecular-weight fractions of the Superose 6 column. As indicated in panel C, the Superose 6 column fractions were combined as the high-molecular-weight pool (H, fractions 6 to 12) and the low-molecular-weight pool (L, fractions 18 to 24). One hundred micrograms of proteins per pool was immunoprecipitated with polyclonal anti-V5 antibody and analyzed by WB, as shown on the right. (E) Exogenous MDMX and S2 are cofractionated with p21 in double-thymidine-arrested cells. GFP-MDMX and V5-S2 were ectopically expressed in H1299 cells. The transfected cells were synchronized in G₁/S phase by a double-thymidine arrest. A 10 μM concentration of MG132 was added to the culture in the second arrest. The cells were harvested and lysed. The cell lysate (300 μg) was subjected to size exclusion chromatography after clarification as described for Fig. 5A. The selected fractions (30 μl/fraction) were revolved by 12% SDS-PAGE followed by WB, as indicated on the right.