Iblot 2 dry transfer device

Western blot was performed as we previously described with some modifications [35 (link)]. Briefly, protein extracts were denatured at 95 °C for 5min in NuPAGE LDS sample buffer (Invitrogen) supplemented with NuPAGE sample reducing agent (Invitrogen). Protein extracts were loaded (25 μg) into 4–12% Bis-Tris gels (Invitrogen), electrophoresed at 200 V for 30 min and electroblotted to PVDF membranes using an iBlot-2 dry transfer device (Invitrogen). Membranes were blocked in PBST containing 5% of BSA and then incubated with primary and respective horseradish peroxidase-conjugated secondary antibodies. Detection and quantification were performed as described in section 2.5. Membrane Coomassie staining was used as loading control. Primary Antibodies: anti-Hsp60 (1:1000, sc-13115), anti-Hsp70 (1:1000, sc-66049), Hsp90 (1:1000, sc-13119), anti-SOD2 (1:500, sc-137254) and TFAM (1:750, sc-23588) from Santa Cruz Biotechnology; anti-SDHA (1:2000, ab14715) from Abcam; anti-SQSTM1/p62 (1:500, #5114) and anti-LC3A/B (1:1000, #4108) from Cell Signaling Technology; and anti-Hsp40 (1:1000, PA5-17382) from Invitrogen. Secondary Antibodies: horseradish peroxidase-conjugated anti-mouse (1:4000, G-21040) and anti-rabbit (1:4000, G-21234) from Invitrogen.