Figure 5

Association with proliferating cell nuclear antigen (PCNA) and Intracellular distribution of SMCX (Smcy homolog, X-linked (mouse)) PCNA-interaction protein motif (PIP box) mutants. (A) Intracellular distribution of PIP mutants. 293T cells were transfected with plasmid encoding SMCX and mutated SMCX Two days after transfection, cells were harvested, then the chromatin fraction was separated from the rest of the lysate and both fractions analyzed by western blotting. SMCX mutants in chromatin: SMCX = normal SMCX, F1209A = F to A mutant (see text), L1204A = L to A mutant, Q1201A = Q to A mutant. H3 and GAPDH served as loading controls. Ch = chromatin fraction, W-Ch = cell lysate without chromatin (bottom), (B) Association of PIP mutants with PCNA 293T cells were transfected as in (A). Two days after transfection, cells were harvested, whole-cell lysates prepared and immunoprecipitated with PCNA. SMCX and PIP mutants in immunoprecipitates were detected using western blotting. Terminology as in (A).