Nsmase2

Protein extracts were subjected to immunoblot analysis using antibodies against Alix, p-ERK, ERK (Cell Signaling Technology, Danvers, MA, USA), GAPDH (Santa Cruz Biotechnology, Dallas, TX), Rab27a (Proteintech, Chicago, IL, USA) and nSMase2 (Santa Cruz Biotechnology). Immune complexes were detected with appropriate secondary antibodies from Santa Cruz Biotechnology and chemiluminescence reagents (Pierce, Rockford, IL, USA) as we described^{54 (link)}. Immunoblot signals were captured using the Image Quant Las 300 (GE Healthcare, Piscataway, NJ, USA). Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD, USA, http://imagej.nih.gov/ij/).

For Western blotting analysis, cells were lysed in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween20, 10 mM β-glycerophosphate) with 1% Protease inhibitor cocktail (Nacalai Tesque). The protein concentration was determined using a Pierce™ BCA Protein Assay Kit (23225, Thermo Fisher Scientific), and the proteins were transferred to a PVDF membrane (EMD Millipore) after SDS-PAGE. Primary antibodies used in this study were anti-SMS2 (sc-366682, Santa Cruz), nSMase2 (sc-166637, Santa Cruz), H-Ras (sc-29, Santa Cruz), Alix (12422, Proteintech, Rosemont, IL, USA), Rab27a (17817, Proteintech), Caspase3 (9662, Cell Signaling Technology), Caspase1 (sc-1780, Santa Cruz), ATG5 (12994, Cell Signaling Technology), p62 (PM045, MBL International, Woburn, MA, USA), LC3 (2775, Cell Signal Transduction), α-tubulin (T9026, Sigma-Aldrich). After incubation with secondary antibodies (GE Healthcare, Chicago, IL, USA), membranes were treated with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and detected by FUSION SOLO S (Vilber Lourmat, Collegien, France).