Anti timm23

The primary antibodies used include anti-LC3B, anti-beclin1 (Novus Biologicals, Littleton, CO, USA), anti-caveolin1 (Abcam, Cambridge, UK), anti-TIMM23, anti-phospho-caveolin1 (BD Bioscience, San Jose, CA, USA), anti-HA, anti-Vps34 (Cell Signaling Technology, Danvers, MA, USA), anti-tubulin, anti-Flag (Sigma-Aldrich), anti-p62 (Abnova, Taiwan) and anti-TOMM20 (Santa Cruz Biotechnology, Dallas, TX, USA). Immunoprecipitation assays were performed as previously described;^{6 (link)} briefly, cells were lyzed with modified RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1 mM Na₃VO₄, 1% CHAPS). After pull-down with the appropriate antibodies, same amounts of protein were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto the polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Immunoblot analysis was then performed and visualized by the enhanced chemiluminescence method.

Western blot was performed as described in our previous study 27 . Antibodies employed in this study were: anti-TRIP-Br1 (Enzo Life Sciences, ALX-804-645), anti-PARP (Cell Signaling Technology, #9542S), anti-cytochrome c (Santa Cruz Biotechnology, sc-7159), anti-CypA (Enzo Life Sciences, BML-SA296), anti-LC3 (Cell Signaling Technology, #2775S), anti-VDAC1 (Cell Signaling Technology, #4661), anti-TOMM20 (BD biosciences, 612278), anti-TIMM23 (BD biosciences, 611222), anti-HSP60 (Santa Cruz Biotechnology, sc-139661), anti-HSP70/GRP75 (Santa Cruz Biotechnology, sc-13967), anti-cathepsin B (Bioworld, BS3536), anti-cathepsin D (Bioworld, BS90201), anti-LAMP1 (Santa Cruz Biotechnology, sc-20011), anti-rabbit (Cell Signaling Technology, #7074S), and anti-mouse (Santa Cruz Biotechnology, sc-516102). Antibodies against γ-tubulin (Santa Cruz Biotechnology, sc-7396) and β-actin (Santa Cruz Biotechnology, sc-47778) were used to measure levels of γ-tubulin and β-actin as loading controls. Results of western blot analysis were semi-quantified using ImageJ software (ver. 1.51u; National Institutes of Health, USA). The relative intensity was compared to γ-tubulin or β-actin level and presented as bar graphs.