Figure 3. VCP/p97 inhibition prevents UV-induced RNAPII proteolysis from chromatin.
(A) CS1AN+iCSB cells were Dox-induced, UV irradiated, maintained for indicated time periods, and then subjected to cellular protein fractionation protocol. RNAPII, VCP/p97, Actin, Lamin B, and ORC2 and PCNA in fractions containing the same amount proteins were detected by Western blotting. Actin, Lamin B and nuclear protein ORC2 served as fractionation mark or as loading controls. “L. Exp.” indicates longer exposure. (B) The CS1AN+iCSB cells were induced for CSB expression and UV-irradiated as in Figure 3A, but were treated with DBeQ. The chromatin fractions were analyzed by Western blotting for RNAPII, ORC2 and VCP/p97. (C) HCT116 cells were pre-treated with 10 μM DBeQ or vehicle for 4 h, UV-irradiated at 50 J/m2 and kept under DBeQ treatment for additional 2 h. Nucleoplasmic, soluble chromatin and insoluble chromatin fractions were isolated by cellular protein fractionation. The RNAPII, 19S proteasomal Sug1 and S1, as well as UBXD7 were examined in indicated chromatin fractions containing the same amount of proteins. Lamin B served as a fractionation mark and a loading control.