Immobilion western chemiluminescent hrp substrate

Cells were lysed with RIPA buffer composed of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS containing protease inhibitor cocktail (Roche, USA) and Halt^TM phosphatase inhibitor (Thermo Scientific, USA). The lysate was heated in 5x protein sample buffer at 100°C for 15 min, separated by 10% SDS-PAGE, and transferred onto PVDF membranes (Millipore Corporation, USA). The membrane was blocked by 5% (w/v) bovine serum albumin (Bioworld, USA) in PBS containing 0.05% (v/v) Tween 20 (PBST) for 1 h and incubated overnight at 4°C with the primary antibody against STAT6 and phospho-STAT6 (1:1000; rabbit monoclonal IgG). Following the addition of horseradish peroxidase (HRP)-labeled secondary antibodies (Santa Cruz Biotechnology, USA) for 1 h, the blots were visualized using Immobilion Western Chemiluminescent HRP Substrate (Millipore Corporation).

Total proteins were extracted using RIPA buffer (ThermoFisher Scientific, #89900, CA, USA), containing phosphatase inhibitor (Roche, #4906845001, Penzberg, Germany) and protease inhibitor (Roche, #4693159001, Penzberg, Germany), separated by SDS-PAGE, and transferred to NC membranes using Iblot 2 NC mini stacks (Invitrogen, #IB23002, CA, USA). Following primary antibodies were used: anti-Glucose-6-phosphate isomerase (Abcam, #ab66340, MA, USA), and anti-GAPDH (Cell signaling technology, #2118, MA, USA); the antibodies were diluted 1:500~5,000 with TBST (Biosesang, #HT2007, Seongnam, Korea) containing 5% skimmed milk. The signals were detected by Immobilion western chemiluminescent HRP substrate (Millipore, #WBKLS0500, Darmstadt, Germany).

Cells were lysed with ice-cold Radio-Immunoprecipitation Assay (RIPA) buffer (150 mM NaCl, 50 mM Tris pH 7.4, 1% NP-40, 0.5% Na-Deoxycholate) supplemented with fresh Protease Inhibitor Cocktail (Sigma) and PhosSTOP (Roche, Rotkreuz, Switzerland) and incubated for 40 min on ice. Cellular debris was pelleted with centrifugation at 2500 g for 10 min at 4°C and the protein concentration was determined by DC-Protein Assay, following the manufacturer's guidelines (Bio-Rad, Cressier, Switzerland). Cell extracts (20–40 µg protein) were resolved by SDS-PAGE on 10 or 12% gels and electrophoretically transferred to PVDF membranes (Immobilion-P, Millipore). Nonspecific binding sites were blocked for 1 h at room temperature with 5% powdered milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBS-T). Blots were then incubated overnight at 4°C with the primary antibody diluted in 5% powdered milk in TBS-T. After washing the blots with TBS-T, secondary antibody diluted in 5% powdered milk in TBS-T was applied for 1 h at room temperature and the antibody binding was detected with Immobilion Western Chemiluminescent HRP substrate (Millipore) or CDP-Star (Roche) and visualized with FujiFilm Las-4000 camera (GE-Healthcare) according to the manufacturer's instructions. Image-J software (National Institutes of Health, Bethesda, MD) was used for densitometric analysis of the Western blots.

To analyze MTUS1 protein expression in cell lines, immunoblotting was performed with 30 μg total protein of whole cell lysates after SDS-PAGE on 7.5% PAA-gels on nitrocellulose membrane using wet blotting method with Mini Protean® Tetra System (BioRad Laboratories, Munich/Germany) according to manufacturer’s protocol. Membranes were blocked with Immunoblot Blocking Reagent (Millipore, Billerica/USA) and treated with anti-MTUS1 antibody (mouse IgG clone 1C7, Abnova H00057509-M01, 1:130, 1 hour/RT, contains epitopes against ATIP1 (49 kDa), ATIP3 (140 kDa) and ATIP4 (59 kDa)) or β-AKTIN (mouse, Sigma-Aldrich, Taufkirchen/Germany, A5441, 1:10 000, 1 hour, RT) and HRP-conjugated secondary antibody (goat-anti-mouse, Dianova/Jackson ImmunoResearch Laboratories, Baltimore/USA, 40 min, RT). Luminescence signal detection was performed using Immobilion Western Chemiluminescent HRP Substrate (Millipore) according to manufacturer’s instructions with Fusion FX7 (Vilber-Lourmat, Eberhardzell/Germany). Cell lysates of LNCaP were included as positive control.

For total protein extraction, cells were washed with cold PBS and lysed in SDS lysis buffer. The lysates were sonicated at low intensity and centrifuged at 12,000 rcf for 2 min at 4 °C. The supernatant containing total cell extract was quantified using the BCA protein assay (Thermo Fisher Scientific Inc.). Equal amounts of protein were run in SDS-PAGE on 10% Bis–Tris NuPAGE gels, transferred to PVDF membranes (Thermo Fisher Scientific Inc.), blocked in 5% BSA and incubated with primary and secondary antibodies diluted in 3% BSA-0.1% Tween-20 in PBS. The primary antibodies used for immunoblotting were: mouse monoclonal γH2AX (1:1000; Millipore), rabbit polyclonal H4K16Ac (1:1000; Active Motif), rabbit monoclonal MOF (1:1000; Abcam) and rabbit polyclonal H3 (1:15,000; Abcam). Primary antibody incubation was performed overnight at 4 ºC. For H3 detection, membranes were incubated with 1:15,000 primary antibody for 1 h at RT. Anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase (Millipore) were used at a dilution of 1:5000 and incubated for 1 h at RT. Peroxidase activity was detected with Immobilion Western Chemiluminescent HRP Substrate (Millipore). Images were acquired with the ChemiDoc XRS + system using Quantity One software (Bio-Rad).

Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere [41 (link)]. Antibodies against p-Rb^ser780, p-Rb^{ser807/ser811}, Rb, Cyclin D1, p16^INK4a, p-CDK6, CDK6, p-AKT^ser473, AKT, pEGFR^tyr1068, EGFR, p53, p-MDM2, p21, c-Myc, actin, and HRP-conjugated secondary antibodies was from Cell Signaling Technology; the chemiluminescence system (Immobilion^TM Western Chemiluminescent HRP Substrate) was from Millipore (Temecula, CA, USA). Reagents for electrophoresis and blotting analysis were from BIO-RAD (Hercules, CA, USA). The whole Western blots are shown in Figures S3–S5.