Journal: Nucleic Acids Research

Article Title: BTG3-dependent VCP/p97 nuclear translocation is required for efficient repair of UV-induced DNA lesions

doi: 10.1093/nar/gkaf626

Figure Lengend Snippet: Loss of BTG3 leads to reduced mobility of XPC at damage sites. ( A – C ) Colocalization of BTG3 and XPC at damage sites after UV. PLA was conducted with HaCaT cells using anti-BTG3 and anti-XPC (A). Mean fluorescence intensity and PLA foci number were quantified and shown in panels (B) and (C), respectively, with n ≧ 100. Data were analyzed using unpaired two-tailed t -test and presented as mean ± SEM Scale bar, 10 μm. ( D ) Co-immunoprecipitation of endogenous BTG3 and XPC from HaCaT cells. ( E, F ) Retention of XPC at localized UV damage in BTG3 KO cells. Localized UVC-irradiation (100 J/m 2 ) was performed with 5 μM isopore membrane filter and XPC loading at localized UV damage sites was assessed using confocal microscopy (E). Scale bar, 10 μm. Percent cells with XPC-positive foci were counted and presented as mean ± SEM in panel (F). Data were analyzed using unpaired two-tailed t -test. n ≧ 50. ( G, H ) Mobility of XPC was reduced in BTG3 KO cells. The mobility of XPC-GFP in mock-treated or global UV-irradiated (20 J/m 2 ) parental and BTG3 KO HEK293T cells was assessed using FRAP. Fluorescence recovery was monitored over 120 s and normalized to prebleach intensity ( n = 36 from 3 independent experiments) (G). Data were analyzed using unpaired two-tailed t -tests and shown as mean ± SEM in panel (H).

Article Snippet: After incubation with PLA blocking solution, HaCaT cells were incubated with anti-XPC (Santa Cruz, 1:200 dilution) and anti-BTG3 (Lab raised, 1:100 dilution) antibodies overnight at 4°C with gentle rocking.

Techniques: Fluorescence, Two Tailed Test, Immunoprecipitation, Irradiation, Membrane, Confocal Microscopy