FIG. 2.

hSNF5 depletion does not significantly affect the XPC level or its modification and does not influence XPC recruitment to the UV damage site. (A) Depletion of hSNF5 protein after hSNF5 siRNA treatment. HeLa cells were transfected with 33 or 66 nM hSNF5 siRNA or a nontargeting control siRNA or mock treated (no transfection). At 48 h after the transfection, lysates were isolated. A Western blot assay was performed with anti-hSNF5 and antiactin antibodies. (B) hSNF5 depletion does not significantly affect XPC levels and ubiquitination. HeLa cells were transfected with 60 nM siRNAs. After 48 h of transfection, cells were treated with 20 J/m² UV or left untreated and harvested at the indicated time points postirradiation. A Western blot assay was performed using anti-XPC, anti-hSNF5, and antilamin antibodies. (C) hSNF5 depletion does not affect XPC focus levels at the UV damage site. HeLa cells were transfected with 60 nM siRNAs. After 48 h of transfection, cells were subjected to UV irradiation (100 J/m²) through micropore filters, left untreated, allowed to repair (1 h), and processed for immunofluorescence using anti-XPC antibody as described in Materials and Methods. The XPC foci in hSNF5- and control siRNA-treated cells were quantitated as follows. An average of 200 merged nuclear foci on five independent microscopic fields were used for the quantitation of focus formation for each experiment. The graph shows the average number of XPC focus-containing cells from at least three independent experiments. The error bars indicates means ± standard deviations. (D) hSNF5 depletion does not affect XPC recruitment at the damage site. Experiments were done as described for panel C. Immunofluorescence was done using anti-XPC antibody. Images from a representative experiment are shown.