Supersignal west pico chemiluminescence substrate kit

Equal amounts of cellular proteins (10 or 15 μg) prepared from MJ and HH cells transduced with INSL3 shRNAs or non-target shRNA lentiviral transduction particles were separated by 4–12% SDS-PAGE gel and electro-transferred onto nitrocellulose membranes. The membranes were blocked with 5% BSA in TBST (Tris-buffered saline, 0.1% Tween 20) for 1 h at room temperature, then incubated overnight with primary antibodies at 4 °C overnight. The primary antibodies and dilutions used were listed in Table S6. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using the SuperSignal West Pico Chemiluminescence Substrate kit (Thermo, Rockford, IL, USA). The equivalent loading of proteins in each well was confirmed by using pan-actin [26 (link)].

Whole-cell protein extracts were prepared from briefly cultured RCAS-PDGFb (≤ 7 passages) and GL261 glioma cells or primary naïve astrocytes using RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) containing EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany). Protein concentration was determined by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and 20 µg of total protein of each sample was resolved on a 10% SDS-PAGE gel, followed by wet transfer of resolved proteins onto a PVDF membrane (GE Healthcare, Chicago, IL, USA). Membranes were blocked and followed by overnight incubation at 4 °C for murine GPNMB (goat, AF2330; R&D Systems, Minneapolis, MN, USA) and for murine GAPDH (mouse, ab8245; Abcam, Cambridge, UK). Membranes were incubated with a secondary for anti-rabbit HRP antibody at 1:2000 (#7074; Cell Signaling Technology) and for anti-goat with IRDye 680RD (925-68071; Li-Cor, Lincoln, NE, USA), developed with SuperSignal West Pico Chemiluminescence substrate kit (Thermo Fisher Scientific). Signal was detected by Molecular Imager Gel Doc XR system (Bio-Rad, Hercules, CA, USA).

CCL136 cells were lysed in lysis buffer (20 mM Hepes, 150 mM NaCl, 2 mM EDTA, 1% NP40, pH 7.9) containing protease inhibitors (Roche, Mannheim, Germany). Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). Blocking was done with 1% skimmed milk in TBS for 30 minutes. Subsequently, membranes were incubated overnight at 4°C with primary antibody anti-LC3 (mouse, diluted 1 : 3000; Nanotools, Teningen, Germany). Horseradish peroxidase-conjugated goat anti-mouse antibodies (Jackson Immuno Research, Suffolk, UK) were used as secondary reagents. For signal detection, the SuperSignal West Pico Chemiluminescence Substrate Kit (Thermo Scientific, Waltham, USA) was used, following the supplier's protocol.