Ab129267

Protein lysates were prepared by scraping cells in 2 × sample buffer (125 mM Tris–HCl, pH 6.8, 20% glycerol, 10% 2-β-mercaptoethanol, 4% SDS, 0.01% bromophenol blue) and boiled at 98 °C for 5 min. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane (0.45 µm, Merck Millipore). Subsequently, membranes were blocked in 2% BSA and incubated with primary antibodies and secondary antibodies conjugated with CF IRDye 680 and 770 (Sigma) for 1 h or overnight. Primary antibodies used were anti-XPA (sc-853, Santa Cruz Biotechnology), anti-SNF2H (ab3749, Abcam), anti-Tubulin (T6074, Sigma), anti-FANCD2 (nb100-316, Novus Biologicals), anti-GFP (ab290, Abcam), anti-SLX4 (NBP1-28680, Novus Biologicals), anti-ERCC1 (ab129267, Abcam), anti-H2B (07-371, Millipore), and anti-RPA70 (2267, Cell signaling). Secondary antibodies were visualized using the Odyssey CLx Infrared Imaging System (LI-COR Biosciences).

mRNA extracts were converted to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Biosystems™, Not. 4368814, Thermo Fisher, Waltham, MA, USA). qPCR was followed separately by using a ERCC1 primer protocol (ERCC1 Human qPCR Primer Pair, NM_202001), OriGene Technologies, Inc., Rockville, MD, USA) and a beta Actin (beta Actin Human qPCR Primer Pair, NM_001101, OriGene Technologies, Inc.) primer protocol. Protein extracts, separated by SDS-PAGE and transferred onto NC membranes (Schleicher & Schuell, Keene, NH, USA), were probed with antibodies against ERCC1 (ab 129267, 1:1000, Abcam, Cambridge, UK), an anti-beta-actin antibody (mAbcam 8226, 1:1000, Abcam), or an anti-myc tag antibody [9E10] (ab32, 1:1000, Abcam). Proteins of interest were detected using peroxidase-conjugated AffiniPure Goat anti-Mouse IgG, Fc Fragment Specific (115-035008, 1:1000, Jackson ImmunoResearch, West Grove, PA, USA) or goat anti-rabbit IgG, F(ab’) 2-HRP (sc-3837, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies, and visualized with the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Merck, Kenilworth, NJ, USA), according to the protocol provided. Quantitation of bands was conducted using Image J.