Fig. 5.

Identification of HERC2 as an XPA ubiquitin ligase. (A) 293T cells were transfected with control Cyclophilin-B siRNA (C), HERC1 siRNA (H1), HERC2 siRNA (H2), or both HERC1 and HERC2 siRNAs (H1/2), and then incubated with cycloheximide (CHX) where indicated and the levels of the target proteins as well as of XPA and GAPDH (loading control) were determined by immunoblotting. (B) Control CycP-B or HERC2 siRNA treated cells were incubated with CHX for the indicated times and the levels of the HERC2, XPA, control proteins, and GAPDH were determined by immunoblots. (C) A549 cell extract was incubated with Protein A agarose and either HERC2 antibodies or control rabbit IgG. The beads were collected and incubated with the indicated proteins at 30 ºC for 30 minutes followed by separation on SDS–PAGE and analysis by western blotting with either Flag antibodies for XPA (Top) or with ubiquitin antibodies (Bottom).