Figure 4. RNAPII interacts with VCP/p97.
(A) U2OS cells, harboring inducible Myc-tagged-Wt-VCP/p97 transgenes (iMyc-Wt-VCP/p97), were transfected with expression constructs for HA-tagged ubiquitin (HA-Ub) for 48 h and in the second 24 h, Dox was added for transgene induction. The transfected cells were UV irradiated and maintained for indicated time. The cell lysates were made in RIPA buffer and subjected to immunoprecipitation. The RNAPII, VCP/p97 proteins in Input (~10%) and immunoprecipitates were detected by Western blotting. Lamin B blot served as the loading control. (B) U2OS cells harboring inducible Myc-tagged EQ-VCP/p97 transgenes (iMyc-Mut-VCP/p97) were used in the experiments similar to that in Figure 4A. (C) HCT116 cells were UV-irradiated at 20 J/m2, and the cell lysates in RIPA buffer were prepared 2 h later. The RNAPII, VCP/p97 and LaminB were detected in Input (~10%) and in immunoprecipitates recovered by RNAPII or control antibody. (D) CS1AN+iCSB cells were Dox-induced for CSB expression for 24 h, UV irradiated at 20 J/m2, maintained for 2 h, and then cell lysates were made in RIPA. The immunoprecipitation and Western blotting were carried out as in Figure 4C. *indicates a modified CSB species.