Fluorchem imaging system

Crude mitochondrial and total fractions were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 12 mM deoxycholic acid, 0.5% Nonidet P-40, and protease and phosphatase inhibitors). Proteins were loaded for SDS-PAGE followed by Western blotting. Nitrocellulose membranes were incubated with primary antibodies and successively with the appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were detected by a Fluorchem Imaging System after incubation of the membranes with ECL Selected Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA).

Crude mitochondria and BAT were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 12 mM deoxycholic acid, 0.5% Nonidet P-40, and protease and phosphatase inhibitors). Protein samples were used for SDS-PAGE followed by Western blotting. Nitrocellulose membranes were stained with primary antibodies against α-Actin, TOMM20, GRP75, H2B (Santa Cruz Biotechnologies, Dallas, TX, USA), vDAC, SDHB, UQCRC2, MTCo1 (Abcam, Cambridge, UK), FoxO1 (Cell Signaling Technologies, Danver, MA, USA), Drp1, OPA1 (BD Transduction Laboratories™, San Jose, CA, USA), SDHA, NDUF8 (MitoSciences, Eugene, OR, USA) all diluted 1:1000. Afterward, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody, and immunoreactive bands were detected by a Fluorchem Imaging System upon staining with ECL Selected Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA). Immunoblots reported in the figures are representative of at least three experiments that gave similar results.

Isolated mitochondria or cell pellets were resuspended in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 12 mM deoxycholic acid, 0.5% Nonidet P-40 and protease inhibitors) or in lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 0.5% IGEPAL CA-630 and protease inhibitors), respectively. Protein samples were used for SDS-PAGE followed by western blotting. Nitrocellulose membranes were stained with primary antibodies against COX4I1 (1 : 500), MFN2 (1.500), TUBB (1 : 1000), PGC-1α (1 : 500), TRX1 (1 : 1000), TOMM20 (1 : 1000), TFAM (1 : 1000), SOD2 (1 : 2000), FOXO1 (1 : 1000), p-RPS6KB1 (1 : 1000), p-PRKAA2 (1 : 1000), PRKAA2 (1.1000), LDH (1 : 5000), PINK1 (1 : 1000), PARK2 (1 : 1000), BNIP3 (1 : 1000), LC3I-II (1 : 000), SQSTM1 (1 : 2000) and Sp1 (1 : 500). Afterward, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody, and immunoreactive bands were detected by a Fluorchem Imaging System upon staining with ECL Select Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA; RPN2235). Immunoblots reported in the figures are representative of at least four experiments that gave similar results. TUBB or TOMM20 were used as the loading control. The possible presence of protein contaminants after isolation of mitochondria was measured by incubating nitrocellulose membrane with anti-H2B.