Confluent fibroblasts were treated in the following conditions: 100 nM Torin (Cell Signaling Technology, Danvers, MA, USA, 14379S) for 3 h at 37 °C, 100 nM Bafilomycin A1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-201550) for 3 h at 37 °C, 100 nM Torin and 100 nM Bafilomycin for 3 h at 37 °C, 20 µM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) (Sigma, C2759) for 24 h at 37 °C, and 20 µM CCCP and 100 nM Bafilomycin for 24 and 3 h, respectively, at 37 °C. Before each treatment fibroblasts were washed with PBS.
Afterward, fibroblasts were washed with PBS, detached using trypsin (Life Technologies, 25300054), and centrifuged at 500× g for 3 min and 30 s. Cell pellets were resuspended in PBS and centrifuged at 1000× g for 5 min at 4 °C. Washed pellets were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% (v/v) NP-40, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholate, pH 8.0) with freshly added protease inhibitors (Merck, 535140), and incubated for 1 h at 4 °C while constantly spinning. Cell lysates were centrifuged at 15,000× g for 15 min at 4 °C, and supernatants were transferred to clean tubes. Protein concentration was determined with a Bradford assay.
Afterward, fibroblasts were washed with PBS, detached using trypsin (Life Technologies, 25300054), and centrifuged at 500× g for 3 min and 30 s. Cell pellets were resuspended in PBS and centrifuged at 1000× g for 5 min at 4 °C. Washed pellets were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% (v/v) NP-40, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholate, pH 8.0) with freshly added protease inhibitors (Merck, 535140), and incubated for 1 h at 4 °C while constantly spinning. Cell lysates were centrifuged at 15,000× g for 15 min at 4 °C, and supernatants were transferred to clean tubes. Protein concentration was determined with a Bradford assay.