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Cells were lysed in RIPA (50 mM Tris HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% NP 40; 0.1% SDS; 0.5% sodium deoxycholate) buffer with protease inhibitor (Roche, Basel, Switzerland). The protein lysate concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin (BSA) as standard. Then, equal amounts of protein samples were separated on SDS-polyacrylamide gels and blotted onto a PVDF membrane (Millipore, Darmstadt, Germany). The following antibodies were used: an anti-Myc tag antibody (1:1000, MA1213161MG, Thermo Fisher Scientific), an anti-Flag M2 antibody (1:3000, F1804, Sigma-Aldrich), an ant-β-actin antibody (1:5000, Ab8226, Abcam, Cambridge, UK), and a chicken anti-mouse IgG-HRP (1:5000, sc-2954, Santa Cruz Biotechnology, Dallas, TX, USA). Immunoblots were visualized with a chemiluminescence detection system (ImageQuant LAS 4000, GE Healthcare Bio-Sciences, Piscataway, NJ, USA).

Whole-cell lysates were extracted with cell culture lysis buffer (Promega, Madison, WI, USA), and protein concentrations were quantified with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. The transfer membrane was blocked and probed with the following antibodies prepared in 5% BSA (Sigma-Aldrich) overnight at 4 °C: anti-STMN1 (1:1000, 13655S, Cell Signaling, Danvers, MA, USA), anti-FLAG-M2-HRP (1:1000, A8592, Sigma-Aldrich), and anti-β-actin (1:5000, A5441, Sigma-Aldrich). The membrane was then probed at room temperature for 1 h with the corresponding secondary antibodies: bovine anti-rabbit IgG-HRP (1:3000, sc-2370, Santa Cruz Biotechnology, Dallas, TX, USA) and chicken anti-mouse IgG-HRP (1:5000, sc-2954, Santa Cruz Biotechnology). Immunoblots were visualized in an ImageQuant LAS 4000 chemiluminescence detection system (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). All Uncropped blots can be seen in Figure S8.

Protein extracts were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The transferred membrane was blocked and hybridized with the following antibodies in 5% BSA (Sigma-Aldrich) overnight at 4 °C: anti-ALIX (1:1000, 2171S, Cell Signaling), anti-TSG101 (1:1000, EXOAB-TSG101-1, System Biosciences), anti-CD81 (1:1000, GTX101766, Genetex), anti-Numb (1:1000, ab14140, Abcam), anti-β-catenin (1:1000, 610154, Becton-Dickinson), anti-CXCL1 (1:1000, AF-453-NA, R&D Systems Inc.), anti-CXCL2 (1:1000, AF-452-NA, R&D System Inc.), anti-FLAG tag (1:1000, F1804, Sigma-Aldrich), and anti-β-actin (1:5000, A5441, Sigma-Aldrich). The corresponding secondary antibodies were used for hybridization at room temperature for 1 h: bovine anti-rabbit IgG-HRP (1:3000, sc-2370, Santa Cruz Biotechnology), chicken anti-goat IgG-HRP (Catalog sc-2953, Santa Cruz Biotechnology), and chicken anti-mouse IgG-HRP (1:5000, sc-2954, Santa Cruz Biotechnology). Immunoblots was visualized with a chemiluminescence detection system (ImageQuant LAS 4000, GE Healthcare Bio-Sciences).