Select western blotting detection reagent

SDS-polyacrylamide gel electrophoresis and immunoblot analysis were performed using protein samples harvested with RIPA buffer (50 mM Tris/HCl pH 8.0, 0.1% SDS, 150 mM NaCl, 1% IGEPAL CA 630, 0.5% deoxycholate) or samples eluted from Anti-FLAG M2 magnetic beads (Sigma-Aldrich). For protein quantification, the Pierce Detergent Compatible Bradford Assay Reagent (Thermo Fisher Scientific) was used. All antibodies were used at the indicated dilutions in 50 mM Tris [pH 7.2], 150 mM NaCl with 0.2% Tween-20, and 5% skim milk powder. Amersham ECL Prime or Select Western Blotting Detection Reagent (GE Healthcare) in combination with the Fusion FX-6 Edge system (Vilber Lourmat) was used for visualization. All antibodies used in this study are listed in Supplementary Data 6. Protein bands detected with the Fusion FX-6 Edge system (Vilber Lourmat) using the Evolution-Capt Edge software (version 18.05) were quantified in a semi-automated manner using the ImageQuant TL 1D software (version 8.1) with a rolling-ball background correction. The control condition was set to unity, quantification results are shown as data points and mean.

Immunoblotting was performed as previously described^{55 (link)}. Briefly, whole cell extracts were obtained by incubating the cells in lysis buffer supplemented with protease and phosphatase inhibitor cocktail. Lysates concentration was determined and equal amounts of proteins were loaded on a 4–12% precast gel (Thermo Fisher Scientific) and transferred to nitrocellulose membranes. Blots were blocked with TBST 5% non-fat dry milk (Bio-Rad Laboratories, Hercules, CA, USA) and incubated overnight at 4 °C with primary antibodies (described in the Antibodies and Reagents section) then incubated for 45 min with secondary HRP-conjugated antibodies dissolved in TBST/1% BSA. Chemiluminescent signals were detected with Amersham ECL prime or select western blotting detection reagent (GE Healthcare Life Sciences, Barrington, IL, USA). Images were taken and analyzed with Bio-Rad ChemiDoc Imagers (Bio-Rad Laboratories).

Protein was extracted from M0505 and STOSE cells using M-PER mammalian protein extraction reagent (GE Healthcare). Tumor tissue from SCA1+ and SCA1− tumors was homogenized and protein was extracted using M-PER mammalian protein extraction reagent. Protein extracts were run on a precast Nupage 4–12% bis–tris gradient gel (Life Technologies) and transferred to a nitrocellulose membrane. Following 1 h blocking in 5% non-fat milk, membranes were incubated with mouse monoclonal PAX8 (1:500, Santa Cruz Biotechnology) or mouse monoclonal P53 (1:1000, Cell Signaling) overnight at 4°C. Following washing, the membranes were incubated with rabbit anti-mouse IgG–HRP (1:5000, Abcam) for 1 h and developed using Select™ western blotting detection reagent (GE Healthcare). The same protocol was used for β-actin using mouse monoclonal anti-β-actin (1:40,000, Sigma Aldrich) and rabbit anti-mouse IgG-HRP (1:15,000, Abcam).