Immobilon western hrp substrate

The GST and GST fusion proteins (0.3 µg) were separated by SDS-PAGE (11% polyacrylamide) and transferred to nitrocellulose membranes (cat. no. 1620112, Advantec Toyo Kaisha, Ltd.). The membranes were blocked with blocking solution [0.5% skim milk powder in a buffer consisting of 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.1% Tween-20], and subjected to specific primary antibodies, i.e., 1:5,000-diluted antibodies against GST (cat. no. 600-101-200, Rockland Immunochemicals, Inc.) or 1:1,000-diluted sera from subjects. Following incubation with 1:30,000-diluted horseradish peroxidase (HRP)-conjugated secondary antibodies [donkey anti-goat (cat. no. sc-2056), or anti-human IgG (cat. no. sc-2453); both from Santa Cruz Biotechnology, Inc.], immunoreactivity was measured using Immobilon Western HRP Substrate (Merck KGaA) and LuminoGraph II (ATTO Co, Ltd.). The results were detected as previously described (21 (link),23 (link)).

Total protein was lysed in a buffer containing 25 mM Tris/NaOH (pH 7.6), 150 mM NaCl, 35 mM sodium dodecyl sulphate (SDS), 12 mM sodium deoxycholate, additionally supplemented with Triton x-100 (1.0%), proteinase inhibitor (complete tablets Mini EDTA-free, Roche, Basel, Switzerland), and phosphatase inhibitor cocktail-2 (Sigma Aldrich), diluted 3:4 in 4× sample buffer containing 14 mM SDS, 200 mM Tris/NaOH (pH 6.8), 10 mM dithiothreitol further containing glycerol (20%) and bromophenol blue (0.005%), and heated (5 min, 70 °C). Proteins were separated by SDS-polyacrylamide gel (10%) electrophoresis, blotted, and probed against ALDH1A3 and GAPDH using primary rabbit polyclonal l anti-ALDH1A3 antibody (#PA5-29188, batch number TH2617862, 1:1000, Thermo Scientific, Waltham, MA, USA), and mouse monoclonal anti-GAPDH antibody (clone 6C5, #ab8245, 1:20,000, Abcam, Cambridge, MA, USA), respectively. Antibody binding was detected after binding of horseradish peroxidase (HRP)-linked antirabbit IgG (#70749, 1:2000, Cell Signaling Technology, Frankfurt a. Main, Germany) and antimouse IgG (#NA931V, 1:2000, GE Healthcare, Buckinghamshire, UK) secondary antibody, respectively, with Immobilon Western HRP substrate (Merck Millipore, Darmstadt, Germany) and recorded with ChemiDoc Imaging System (BioRad Laboratories, Feldkirchen, Germany).

One 75 cm² flask of cells at ~90% confluence was harvested by scraping and centrifugation (1000 × g, 5 min), washed twice with ice-cold PBS, resuspended in 300 to 500 μl lysis buffer (50 mM Tris pH 7.4, 0.1 M NACl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF, cOmplete inhibitors) for 30 min on ice, clarified by centrifugation (10,000 × g, 10 min at 4 °C), and protein concentration was determined (BCA assay, Thermo Scientific). Lysate concentrations were normalised, and the lysates were heated in NuPAGE SDS sample buffer containing 10% β-mercaptoethanol at 90 °C for 5 min, run on a gel and transferred to nitrocellulose. All blots were blocked in 5% (w/v) milk in PBS-T (PBS with 0.1%(v/v) Tween-20) for 1 h, incubated overnight at 4 °C with primary antibody in the same blocking solution (Supplementary Table 2), washed three times with PBS-T for 5 min, incubated with a 1:3000 dilution of species-specific HRP-conjugated secondary antibody (GE Life Sciences) in 0.1% (w/v) milk in PBS-T for 1 h, washed five times with PBS, and detected with Immobilon Western HRP substrate (Merck).

The total proteins from each sample were separated by electrophoresis on 8% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (IPVH00010, Merck Millipore, USA). After being blocked with 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4° C overnight (Supplementary Table 2). Then, the membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000, E-AB-1003, Elabscience, China) for 1 h at room temperature. The proteins on the membranes were visualized with Immobilon Western HRP Substrate (WBKLS0500, Merck Millipore) and scanned on a SuperSignal chemiluminescent detection system (BioRad, USA).

Roswell Park Memorial Institute-1640 medium (RPMI-1640; Cat. No. 043–30085), Dulbecco’s modified Eagle’s medium (DMEM; Cat. No. 189–02025), and non-essential amino acids (NEAA; Cat. No. 139–15651) were purchased from Wako Pure Chemical Industries (Osaka, Japan). SAS (Cat No. S0883) and 2’,7’-dichlorofluorescin diacetate (DCFDA; Cat. No. D6883), siRNA (Product no. Mm_Slc7a11_1916), and MISSION siRNA Universal Negative Control #2 (Product no. MISSSION_SIC-002) were purchased from Sigma-Aldrich (St. Louis, MO). Lipofectamine^® RNAiMAX and Opti-mem were purchased from Thermo Fisher Scientific (Waltham, MA). Water soluble tetrazolium salt-1 (WST-1; Cat. No. W201) and 1-methoxy phenazine methosulfate (Cat. No. M003) were purchased from Dojindo (Kumamoto, Japan). The following antibodies were used for western blotting and immunostaining: anti-γ-tubulin (Cat. No. ab179503, Abcam, Cambridge, United Kingdom), anti-pSer139-histone H3 (γH2AX; Cat. No. #9718, Cell Signaling Technology, Beverly, MA), Alexa Fluor 488- or 598-conjugated secondary antibodies (Thermo Fisher Scientific), anti-xCT (Cat. No. sc-98552), anti-actin (Cat. No. sc-1615), and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Immobilon western HRP substrate was purchased from Merck Millipore (Billerica, MA).