Sc 263

We performed immunoblot analyses as published previously [20 , 54 (link)]. In brief, we lysed the cells using RIPA buffer (Merck KGaA, Darmstadt, Germany). We used 10% polyacrylamide gels and transferred the proteins onto a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, Waltham, MA, US). Blots were blocked in Tris-buffered saline containing 5% skim milk (Beckton, Dickinson & Company, Franklin Lakes, NJ, US). Primary antibodies were applied over night at 4 °C, secondary antibodies for 1 h at room temperature. We detected the results documented with a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, US). Subsequent analysis was performed with Image Lab software (Bio-Rad, Hercules, CA, US). We used the p53 antibody Sc-263 (Santa Cruz, Dallas, TX, US), MGMT ab39253 (Abcam, Cambridge, UK) and GAPDH 2118 (Cell Signaling, Technology, Danvers, MA, US). As secondary antibodies we used goat pAb to mouse IgG (HRP), ab97023 (Abcam, Cambridge, UK) and goat pAb to rabbit IgG (HRP) ab97051 (Abcam, Cambridge, UK).

Cells were lysed in a lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 2 mM EDTA; 1% NP-40; and 0.1% sodium dodecyl sulfate) that contained a protease inhibitor cocktail (Complete Mini; Roche Diagnostics, Branchburg, NJ). The blots were cutted according to molecular size markings prior to hybridisation with antibodies. The membranes were incubated with antibodies against human Bcl2 (1:200 dilution, sc-7382, Santa Cruz Biotechnology), ABCG2 (1:50 dilution, sc-58,222, Santa Cruz Biotechnology), P53 (1:500 dilution, sc-263, Santa Cruz Biotechnology), P27^Kip1 (1:500 dilution, sc-528, Santa Cruz Biotechnology), LGR5 (1:200 dilution, Abnova, Taipei, Taiwan) and β-actin (1:1000 dilution, sc-47,778, Santa Cruz Biotechnology) at 4 °C overnight followed by incubation with horseradish peroxidase-conjugated secondary immunoglobulin G (IgG; Thermo Fisher Scientific, New York, NY). The membranes were briefly incubated with an enhanced chemiluminescence reagent (Millipore, Billerica, Mass) and then visualized on X-ray films [31 (link)].

Whole cell protein extracts were prepared by cell lysis with SDS-PAGE buffer (80 mM Tris-HCL pH 6.8, 16% glicerol, 4.5% SDS, 450 mM DTT, 0.01% bromophenol blue) with 200U/ml benzonase (Sigma) and 50 µM MgCl₂ and boiling for 5 min. Equal amounts of protein extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Immunoblotting was performed with antibodies against the following proteins: H3K36me3 (ab9050; Abcam), histone H3 (ab1791; Abcam), phospho-53BP1 (phosphoSer1778; No.2675, Cell Signaling, Danvers, MA), phospho-ATM (phosphoS1981; No.200-301-400s, Rockland, Gilbertsville, PA), total ATM (PC116; Millipore), total RPA32 (ab2175; Abcam), phospho-RPA32 (phosphoSer4/Ser8; A300-245A, Bethyl, Montgomery, TX), α-Tubulin (T5168; Sigma); γH2AX (phosphoSer139; 05-636, Millipore); Histone H2B (ab1790; Abcam); GFP (11814460001; Roche, Basel, Switzerland); total p53 (sc-263; Santa Cruz); phospho-p53 (phosphoSer15; ab38497, Abcam); p21 (sc-397; Santa Cruz).