D1m5y

Cells were lysed for 30 min on ice in RIPA buffer (Thermo Fisher Scientific), including protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). For the detection of FANCD2 in HAP1 cells, cells were treated with MMC 500 ng/ml overnight and protein samples were run on a 3–8% Tris-Acetate gel (Thermo Fisher Scientific). Samples were blotted to a 0.45 μm nitrocellulose membrane. Protein samples were run on a 4–12% Bis-Tris gel (Thermo Fisher Scientific) to detect XPC, XPA, XPF and CSB in HAP1 cells. Antibodies used were anti-XPC (D1M5Y, Cell Signaling, 1:1000), XPF (D3G8C, Cell Signalling, 1:1000), XPA (D9U5U, Cell Signalling, 1:1000), CSB (ab96089, abcam, 1:1000) and FANCD2 polyclonal antisera (1:3000) [64 (link)].

Immunofluorescence were performed according to standard protocols with the following antibodies: anti-XPC (1:200, D1M5Y, Cell Signaling), anti-XPA (1:200, ab85914, Abcam), anti-CPD (1:50, KTM53, Kamiya Biomedical Company), anti-TFIIH p62 (1:50, Q-19, Santa Cruz Biotech) anti-DDB-1 (1:100, ab97522, Abcam), anti-DDB-2 (10 µg mL⁻¹, ab51017, Abcam), and anti-ERCC1 (1 µg mL-1, ab2356, Abcam). All slides were cover-slipped using Vectashield-DAPI mounting media (Vector laboratories, Burlingame, CA, USA). Images per sample were captured using the Leica SP5 Confocal Microscope at BU Cellular Imaging Core. To determine the relative fluorescence at local irradiated sites, the CPD-positive area was gated by the Image J software and fluorescence intensity was quantified. Then the fluorescence intensity of XPC at the same area was quantified and divided by the CPD fluorescence. Relative fluorescence was calculated as the experimental fluorescence divided by the control fluorescence^{69 (link)}.