Ab97522

Cell lysates were prepared with high salt lysis buffer (150 mM Tris-HCl [pH 7.9], 500 mM NaCl, 1% (w/v) Nonidet P-40). Immunoblotting was performed according to a standard procedure using the following primary antibodies: anti-GST (sc-138, Santa Cruz Biotechnology), anti-β-actin (ab6276, Abcam), anti-Aiolos (sc-101982, Santa Cruz Biotechnology), anti-GSPT1 (ab49878, Abcam), anti-DDB1 (ab97522, Abcam), anti-CUL4A (ab72548, Abcam), anti-ROC1 (ab86862, Abcam), and anti-CRBN (CRBN65 mAb, Celgene)^{34 (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)}.

Serum samples were separated through SDS-PAGE and were then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After blocking with Block Ace reagent (DS Pharma Biomedical, Osaka, Japan), membranes were incubated with goat polyclonal anti-SNX5 (1:2000, ab5983, abcam, Tokyo, Japan), rabbit polyclonal anti-SRFBP1 (1:2000, ab109598, abcam), rabbit polyclonal anti-DDB1 (1:2000, ab97522, abcam), rabbit monoclonal anti-CPSF6 (1:2000, ab175237, abcam), or mouse monoclonal anti-ACACA (1:2000, ab205883, abcam) antibody. The second western blot was conducted with rabbit polyclonal anti-SNX5 (1:2000, SAB2102260, Sigma-Aldrich, Tokyo, Japan) antibody. Immunoreactive bands were detected using enhanced chemiluminescence (EMD Millipore, Temecula, CA, USA) after incubation with horseradish peroxidase labeled anti-mouse, rabbit, or goat IgG (1:5000, Vector Laboratories, Burlingame, CA, USA). Signals were detected using C-DiGit Blot Scanner (LI-COR) and then band density was assessed with Image Studio DiGit software (version 5.2) [38 (link)]. The whole blot can be found at supplementary materials (Figures S2 and S3).