Protease inhibitor cocktail

Following treatment, cells were lysed in ice cold radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail and PMSF (AppliChem, Darmstadt, Germany). Lysates were boiled in 1× SDS-PAGE sample buffer (25 mM Tris-HCl, pH 6.8; 10% glycerol, 2% SDS, 2.5% β-mercaptoethanol, 0.005% bromophenol blue) and equivalent amounts of protein were electrophoresed on SDS-PAGE gels. PVDF membranes were blotted with antibodies that recognize Akt, (#2938, 1:1000), p-Akt Thr308 (# 2965, 1:2000), p-Akt Ser473 (# 4060, 1:1500), GAPDH (#2118, 1:1000), (all Cell Signaling, Frankfurt, Germany), ATM (# 1549-1), p-ATM S1981 (# 2152-1, Abcam, Cambridge, UK) and subsequently incubated with anti-rabbit/anti-mouse HRP (horseradish peroxidase)-linked IgG antibodies (1:5000, Millipore, Darmstadt, Germany). Proteins were visualized using enhanced chemiluminescence detection system (Amersham, Munich, Germany). Generally, specific expression was normalized to the mean value of the total blot, or, when specified, to the untreated control. Raw data of Western Blots is provided in Figure S7.

Following treatment, cells were lysed in ice cold RIPA buffer supplemented with protease inhibitor cocktail and PMSF (AppliChem, Darmstadt, Germany). Lysates were boiled in 1× SDS-PAGE sample buffer (25 mM Tris-HCl, pH 6.8; 10% glycerol, 2% SDS, 2.5% β-mercaptoethanol, and 0.005% bromophenol blue) and equivalent amounts of protein were electrophoresed on SDS-PAGE gels. PVDF membranes were blotted with antibodies that recognize Rad51 (#ab133534, 1:10,000, Abcam, Cambridge, UK), β-actin (clone AC-15, #A5441, 1:5000, Sigma-Aldrich, St. Louis, MI, USA), and α-tubulin (#sc-5286, 1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and subsequently incubated with anti-rabbit/anti-mouse IgG HRP (horseradish peroxidase)-linked antibodies (1:5000, Millipore, Darmstadt, Germany). For the cell line UD-SCC-2, β-actin was used as loading control, because this cell line did not express α-tubulin. Proteins were visualized using an ECL chemiluminescence detection system (Amersham, Munich, Germany).

The MITF protein in transfected G361 cells was verified by western blot analysis using a mouse monoclonal anti-Flag M2 antibody. Cells cultured for 48 h were harvested and protein isolated using RIPA lysis buffer (Bio-Rad), supplemented with protease inhibitor cocktail (AppliChem, Germany) and 1 mM phenylmethylsulfonyl fluoride (Merck, Germany). The proteins (20 µg) were then subjected to SDS-PAGE (12%) and transferred onto a polyvinylidene fluoride membrane with pore size 0.22 µm. The membrane was blocked using blocking solution (3% bovine serum albumin in TBS plus 0.1% Tween-20) for 2 h and then stained with a mouse monoclonal anti-Flag M2 antibody (1:1,000 dilution, Sigma) at 4 °C overnight. A goat anti-mouse GAPDH antibody (Thermo Scientific) was used as a control for protein loading. After washing with washing buffer (TBS plus 0.1% Tween-20) for 15 min, four times, the membrane was stained for 1 h with a goat anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (1:1,000 dilution, Thermo Fisher Scientific) at room temperature followed by four washes. Protein levels were measured using Luminata Forte Western HRP substrate (Merck, USA) with a C-DiGit Chemiluminescence Western Blot Scanner (LI-COR Bioscience).