Figure 1.
In vivo SUMO modification of human PCNA. (A) HEK293T cells were co-transfected with HA-PCNA, His-UBC9 and either FLAG-SUMO1, or FLAG-SUMO2, or FLAG-SUMO3. In 48 h, post-transfection cells were UV-treated (30 J/m2) or mock-irradiated and, after 3 h lysed and immunoprecipitated on FLAG-beads. FLAG-SUMO precipitates were immunoblotted with anti-HA antibody to detect PCNA and the SUMO-modified forms of PCNA. The lower panel shows the anti-HA western blot of the lysates. (B) Cell lysates and FLAG immunoprecipitates from control HeLa S3 cells and HeLa S3 cells stably expressing FLAG-SUMO1 were immunoblotted with anti-PCNA antibody to detect SUMOylated forms of endogenous PCNA. (C) HEK293T cells were co-transfected with FLAG-SUMO1, His-UBC9 and either HA-PCNA or K164R PCNA and 48-h post transfection cells were lysed and immunoprecipitated on FLAG-beads. FLAG-SUMO1 precipitates were immunoblotted with anti-HA antibody to detect the effect of the K164R mutation on the SUMOylation of PCNA. (D) Interaction of PCNA with UBC9 was tested by co-expressing FLAG-PCNA and HA-UBC9 in HEK293 cells followed by FLAG immunoprecipitation and then by western blot analysis with anti-HA and anti-FLAG antibodies for UBC9 and PCNA, respectively.