Uqcrc2

The Eu was purchased from National Institutes for Food and Drug Control (China). Dulbecco’s modified Eagle medium (DMEM), Nutrient Mixture F-12 (DMEM/F12) media, horse serum, trypsin and penicillin/streptomycin, were obtained from Life Technologies, GibcoBRL (Rockville,MD,USA). Antibodies against CPT-1C and Tomm20 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,USA). Antibodies against ERRα and c-Myc were obtained from Cell Signaling Technology (Beverly, MA,USA) and an antibody against the HRAS, PGC-1β, NDUFB8, SDH-B, UQCRC2, ATP5A, COX5A, MCAD and PPARα were obtained from Abcam (Cambridge, MA, USA). 10058F4 were purchased from Sigma-Aldrich (St.Lois, MO, USA). XF Palmitate-BSA FAO Substrate was obtained from seahorse Bioscience (North Billerica, MA, USA).

The sample for the western blotting was prepared by scraping the cells in PBS. The protein extraction was done using RIPA lysis buffer. The amount of protein was determined using Bradford reagent and spectrophotometer for equal loading of a gel. The samples were prepared in Laemmli loading buffer. 10–20% precast Tricine gel (Invitrogen) was used (particularly for MIC13 as it is a small protein). The proteins were transferred onto nitrocellulose membrane and probed for various antibodies. Anti-HRP secondary antibodies were used. We used following antibodies for immunoblotting, MIC13 (Pineda, Berlin, polyclonal antibody raised in rabbit using following peptide CKAREYSKEGWEYVKARTK), MIC27/APOOL (Atlas Antibodies, HPA000612), MIC26/APOO (Thermo-Fisher, MA5-15493) MIC60/Mitofilin (Pineda, Berlin, polyclonal antibody raised in rabbit against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), MIC10/MINOS1 (Pineda, Berlin, polyclonal antibody raised in rabbits against CQHDFQAPYLLHGKYVK), MIC25/CHCHD6 (cell signaling), VDAC (abcam), β-tubulin (Cell signaling), MIC19/CHCHD3 (abcam), ATP5L (proteintech), ATP5O (abcam), COXIV (abcam), NDUFB4 (abcam), UQCRC2 (abcam), MTND1 (abcam), TOM20 (Proteintech), TIM23 (BD biosciences), human TAZ1 (gift from Steve Claypool). Recording and visualization of chemiluminescent signals were done using VILBER LOURMAT Fusion SL (Peqlab).

The cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (Selleckchem). The protein concentration was tested with a BCA kit, and appropriate amounts of protein were prepared for SDS-PAGE and then transferred to PVDF membrane (Millipore, MA, USA). The membranes were blocked for 1 h with 5% non-fat dry milk and then incubated with rabbit anti-SIRT1 (1:1000; #9475; Cell Signaling Technology Europe, Netherlands); TFAM (1:1000; #8076; Cell Signaling Technology Europe, Netherlands); CATALASE (1:1000; #12,980; Cell Signaling Technology Europe, Netherlands); SOD2 (1:1000; #MA1-106; Invitrogen); UQCRC2 (1:1000; #ab14745; Abcam); MTCO2 (1:1000; #ab110258; Abcam); ASCL4 (1:500; #sc-365230; Santacruz); Actin-b (1:5000, A5441; Sigma) mABs were used as loading controls. The results were imaged using a gel image analysis system (Bio-Rad, California, USA) according to the manufacturer’s instructions.

Immunoblot analysis was used to assess protein levels of subunits of complexes I, III and IV, and subunits of F₀F₁-ATPase in renal cortical mitochondria using 20 μg of mitochondrial protein, as described previously [57 (link),61 (link)]. Antibodies against NDUFA9, NDUFS3, and NDUFB7 subunits of complex I (Cat #A21344, A21343, and 21359) were purchased from Molecular Probes/ThermoFisher Scientific (Waltham, MA, USA). Antibodies against α- and γ-subunits of F₀F₁-ATPase (Cat #ab14748 and #ab119686, respectively) and the core protein 1 (UQCRC1), core protein 2 (UQCRC2), and Rieske protein subunits of complex III (Cat #ab110252, #ab14745, and #ab14746, respectively) were supplied by Abcam (Cambridge, MA, USA). Antibodies against β-subunit of F₀F₁-ATPase (Cat #A21351) were supplied by Life Technologies Corporation (Carlsbad, CA, USA). Cytochrome oxidase subunit I antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat #sc-58347). Citrate synthase levels served as the loading control using the citrate synthase antibody (Cat #D7V8B) supplied by Cell Signaling Technology (Danvers, MA, USA).

The following primary antibodies were used for immunoblotting: NDUFA9 (Molecular Probes, Eugene, OR, USA, A21344), NDUFB8 (Abcam, Cambridge, UK, ab110242), SDHA (MitoSciences, Eugene, OR, USA, MS204), UQCRC2 (Abcam, ab14745), COX1 (Abcam, ab14705) and COX2 (Molecular Probes, A6404), ATP5A (Abcam, ab14748), ATPB (Abcam, ab14730) and TOM20 (Santa Cruz, Heidelberg, Germany, sc11415). HRP-conjugated anti-mouse or anti-rabbit secondary antibodies were used (P0260 and P0399 respectively; Dako, Glostrup, Denmark). Chemiluminescence ECL Prime Kit (Amersham, Little Chalfont, UK) and ChemiDocMP Imaging System (Bio-Rad, Hemel Hempstead, UK) were used for signal detection and Image lab 4.0.1 (Bio-Rad) software for analysis.

The following reagents were purchased: LPS, from Sigma-Aldrich (St. Louis, MO, USA); Cap (purity ≥ 98%), from the National Institutes for Food and Drug Control (Beijing, China); adenovirus pAD/14-3-3γ-shRNA, from Gene Chem Co., Ltd (Shanghai, China); bafilomycin A1 (BafA1) and compound C, from Sigma-Aldrich (St. Louis, MO, USA); antibodies against 14-3-3γ, P62, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 (NDUFB8) and cytochrome b-c1 complex subunit 2 (UQCRC2), from Abcam (Cambridge, UK). Anti-LC3, -AMPKα, -phospho-AMPK (phosphorylation at Ser172), -mTOR, -phospho-mTOR (phosphorylation at Ser2448), -ULK1, -phospho-ULK1 (phosphorylation at Ser757) antibodies, from Cell Signaling Technology (Beverly, MA, USA), and horseradish peroxidase-conjugated IgG secondary antibody, from Zsbio (Beijing, China).

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.