Figure 5

PCNA ubiquitin laddering is not altered by the knockdown of MMS2 and UEV1a. (A) Hela and (C) 293T cells were treated as in Figure 1 followed by immunoblotting for MMS2 and UEV1a (B) Hela and (D) 293T cells were transfected as described in figure 1. Seventy-two hours post-transfection, cells were irradiated with 30 J/m² of UV and lysed 6 h later in boiling SDS, diluted in lysis buffer, and subjected to immunoprecipitation with a PCNA antibody and immunoblotted for PCNA (upper panel) and Ub (lower panel). The controls in the immunoprecipitations were "B+L", in which lysates were incubated with beads but no PCNA antibody, and "1°+B" in which PCNA antibody was incubated with beads alone. Asterisks denote immunoglobulin heavy and light chains as detected in the immunoprecipitations.