Ab124741

Protein expression was determined by immunoblotting. Briefly, cell pellets were prepared in radioimmunoprecipitation assay (RIPA) buffer (Sigma) in the presence of a protease inhibitor cocktail (Roche). Total protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Forty micrograms of protein were electrophoresed on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-FL membranes (Millipore). Membranes were blocked in tris buffered saline with Tween-20 (TBS-T; 50 mM Tris/HCl, 150 mM NaCl, pH 7.5 plus 0.2% Tween-20) and 5% non-fat milk for 1 h at room temperature. Blots were probed with the following primary antibodies: rabbit anti-OGG1 (ab124741, Abcam) at 1/2500 dilution, and mouse anti-β-actin (A5441; Sigma) at 1/10,000 dilution in TBS-T containing 5% non-fat milk. Anti-mouse and anti-rabbit IgG-HRP (immunoglobulin G horseradish peroxidase; Dako) were used as the secondary antibodies, and the immunoblots were developed using Immobilon Classico Western HRP substrate (Millipore). Each immunoblot was performed in triplicate. Images were analyzed using ImageJ software (NIH Image), and OGG1 protein level was normalized to actin levels. The full-length blots are presented in Supplementary Figure S2.

The Human ON-TARGETplus siRNA Library-DNA Damage Response and the siRNA targeting OGG1, PARG, Pol β, and XRCC1, each containing a pool of four different siRNA sequences to help aid knockdown efficiencies, were from Horizon Discovery (Cambridge, UK). The non-targeting control siRNA (AllStars Negative Control siRNA) was from Qiagen (Manchester, UK). The following antibodies were used: γH2AX (05–636; Merck-Millipore, Watford, UK), OGG1, PARG, phosphorylated ATM and phosphorylated DNA-PKcs (ab124741, ab169639, ab81292 and ab18192; Abcam, Cambridge, UK), PARP-1 (sc-53643; Santa Cruz Biotechnology, Heidelberg, Germany), OGG1 for immunofluorescence (NB100-106; Bio-Techne Ltd, Abingdon, UK), Polβ and XRCC1 (kindly provided by G. Dianov) and tubulin (T6199; Sigma-Aldrich, Gillingham, UK). Goat anti-mouse Alexa Fluor 555 or goat anti-rabbit Alexa Fluor 488 secondary antibodies for immunofluorescence were from Life Technologies (Paisley, UK). The OGG1 inhibitor TH5487, as previously described [21 (link)], was used at a concentration of 10 µM. The PARG inhibitor PDD00017273, also previously described [22 (link)], was used at a concentration of 1 µM.

For sample preparation, cells were incubated with 20 µM TH5487 for 2 h at 37 °C, before they were submitted for 3 min at twelve different temperatures ranging from 37 to 62 °C. After the addition of lysis buffer [50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS supplemented with complete protease inhibitor cocktail (Roche)], cell lysis by freezing–thawing took place. In the following centrifugation (30 min at 17,000g at 4 °C) was performed and 70 µL of the supernatant was mixed with 23 µL loading dye before the samples were heated at 95 °C for 10 min. In the following, SDS-PAGE and WB were performed. The membrane was blocked with 5% skimmed milk for 1 h at room temperature. As primary antibodies rabbit anti-OGG1 (ab124741, Abcam) 1:1,000 and mouse anti-Actin (ab6276, Abcam) 1:5,000 were used. This experiment was performed once in U2OS cells (parental) and once in U2OS OGG1-GFP cells (not shown). Data is available.

Various amounts of extracts and purified proteins (Supplementary Figure S2) were loaded onto 4–20% tris-glycine polyacrylamide gels (Invitrogen; XP04202BOX). Proteins were transferred onto a polyvinylidene difluoride membrane followed by blocking in 20% nonfat dry milk (diluted in PBST: phosphate-buffered saline containing 0.1% Tween 20) for 1 h at room temperature. Membranes were incubated with primary antibodies for 2 h at room temperature or overnight at 4°C, washed 3 × 10 min in PSBT, and incubated with peroxidase conjugated secondary antibodies for 1 h at room temperature. Membranes were washed again before developing using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific; #34095). Primary antibodies used: PARP1 (1:100; abcam #ab227244), DDB2 (1:1000; abcam #ab181136), DDB1 (1:1000; Invitrogen #37-6200), XPC (1:1000; Novus #NB100-477) Polβ (1:1000; proteintech #18003-1-AP), OGG1 (1:1000; abcam #ab124741), and APE1 (1:100; Abcam #ab194). Secondary antibodies used: anti-rabbit IgG (1:50,000 Sigma #A0545), or anti-mouse IgG (1:50 000 Sigma #A4416). Blots were analyzed on ImageJ v1.53k. Overexpressed proteins were compared to purified proteins of interest, and in cases of XPC and DNA polymerase β (Polβ) the levels of endogenous protein from nuclear extracts without transfection were utilized (Supplementary Figure S2D).

Cells were washed in cold PBS and lysed in RIPA buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS supplemented with complete protease inhibitor cocktail (04693116001, Roche), freeze-thawed once, incubated 20 min on ice followed by sonication at 80% amplitude, 0.7 cycle and 10 cycles in a UP100H (Hielscher Ultrasonics) and clarification by centrifugation. Proteins were separated and blotted with 4–12% polyacrylamide gels and the Trans-Blot Turbo transfer system, respectively (BioRad). The following primary antibodies were used: rabbit anti-OGG1 (ab124741, Abcam) 1:2500, mouse anti-Actin (ab6276, Abcam) 1:10 000, goat anti-vinculin 1:1000, rabbit anti-histone 3 (ab1791, Abcam) 1:5000, mouse anti-γH2AX(p-Ser139) (05-636, Millipore) 1:2000).