Anti xpc

Whole-cell extracts were prepared by lysing the cells directly in 1 × sodium dodecyl sulphate-polyacrylamide gel electrophoresis sample buffer and subsequent sonification. Antibodies were diluted 1:200–1:1000 in 5% bovine serum albumin (BSA) or 5% milk, 0.1% Tween-TBS and incubated overnight at 4 °C. Anti-p53, anti-HSP90, anti-DDB2, anti-XPC, anti-XPA, anti-ERCC1, anti-XPF, anti-XPG, anti-Pol ε, anti-Pol δ, anti-Lig I and anti-Lig III antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-pChk1, anti p53ser15 and Anti-p53ser46 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The protein–antibody complexes were visualized by Pierce ECL Western Blotting Substrate (32106, Thermo Fisher).

Proteins were analyzed on NuPAGE 4–12% Bis-Tris Protein Gels (Invitrogen) and subjected to immunoblotting analysis. Antibodies used for immunoblotting included anti-cleaved PARP (#9546), anti-cleaved Caspase-3 (#9661), anti-GAPDH (#2118), anti-XPC (sc74410), anti-XPA (sc-853) from Santa Cruz Biotechnology, and anti-RPA32 (NA18, Calbiochem), anti-p62 (sc-292), anti-p89 (sc-293), anti-Caspase-3 (#9665) from Cell Signaling Technology. Secondary antibodies included horseradish peroxidase-linked anti-mouse (sc-2005) and anti-rabbit IgG (sc-2004) from Santa Cruz Biotechnology. Chemiluminescence was visualized with Amersham ECL Western or Prime Western Blotting Detection Reagent (GE Healthcare) on an ImageQuant LAS 4000 Mini apparatus (GE Healthcare) and quantified using ImageQuant TL Software (GE Healthcare). The specificity of all commercially available antibodies used for immunoblotting was verified with appropriate size markers, and the graphed data validates the reproducibility our findings in at least two independent experiments.