Tubulin antibodies

Whole cell extracts (WCE) were prepared as previously described (26 (link),27 (link)). WCE from 20 g HeLa cell pellets (Cilbiotech, Belgium) was fractionated using column chromatography, and proteins present in active fractions from the final Mono-Q chromatography were identified by tandem mass spectrometry, as recently described (27 (link),28 (link)). Following each chromatography stage, protein fractions were analysed for in vitro BER activity using a free or mononucleosome substrate and active fractions pooled for the next chromatography step. Immunoblotting was performed as described in the references above, using the Odyssey Image Analysis System for protein detection and quantification. Primary antibodies raised against APE1 were kindly provided by Prof. G.Dianov, HECTD1 and Mule antibodies were from Bethyl Laboratories (Montgomery, USA), tubulin antibodies were from Sigma (Dorset, UK), Cul4A, DDB1 and histone H2B and H4 antibodies were from Abcam (Cambridge, UK), and histone H3 and H4 antibodies were from Cell Signaling (London, UK).

Cells treated with DMSO or compounds for different duration were lysed using RIPA buffer (Teknova) containing protease and phosphatase inhibitors (Sigma Aldrich). Equal amounts of protein were resolved in 4–12% SDS-PAGE gels (Invitrogen), transferred onto 0.45 µm PVDF membrane (Invitrogen), and incubated with primary antibodies (1∶1000 dilution) overnight. Antibodies for AKT, phospho-AKT (Ser473 and Thr308), phospho-FOXO (Thr24/32), phospho-MEK1/2 (Ser217/221) were purchased from Cell Signaling Technologies. Phospho-GSK3β (Ser9) antibody was from R&D Systems. PRAS40 and phospho-PRAS40 (Thr246) antibodies were obtained from Millipore. Phospho-ERK1/2 (Tyr204), ERK1/2, and phospho-Caspase 9 (Ser196) antibodies were purchased from Santa Cruz Biotechnologies. Tubulin antibodies were purchased from Sigma Aldrich. Following incubation with primary antibody, blots were washed and incubated with IRDye-680 goat-anti-mouse or IRDye-800CW goat-anti-rabbit antibodies (1; 10,000 dilution; LI-COR) for 1 h. Following thorough washing, blots were analyzed using an infrared imaging system (LI-COR).