Phosphatase inhibitor cocktail

HeLa cells and skin fibroblasts were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH=8, 1 mM EGTA, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS), supplemented with Complete EDTA-free protease inhibitor cocktail (Roche Applied Science), and Phosphatase inhibitor cocktail (Roche Applied Science). Following incubation on ice for 30 min, crude extracts were centrifuged at 15000 × g for 10 min at 4 °C to remove debris, aggregates and insoluble proteins. The resulting supernatants, containing the extracted proteins were quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific), and are referred as whole cell lysates. For western blotting, 20 μg of whole cell protein extracts were dissolved in LDS sample buffer (Life Technologies) supplemented with 100 mM dithiothreitol (DTT), and heated for 5 min at 95 °C. Proteins were then separated via SDS-page electrophoresis on 4–12% Bis-Tris NuPage gels (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane (Thermo Fisher Scientific) using semi-dry electrophoretic transfer (Bio-Rad). Membranes were subsequently blocked for 1 h at RT with either 5% non-fat dry milk in TBS-tween (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween) or 5% Bovine Serum Albumin (BSA) (Sigma-Aldrich) in TBS-tween, based on the primary antibody used. The membranes were then incubated with the primary antibody overnight at 4 °C. Secondary HRP-conjugated antibodies, obtained from Bio-Rad, were incubated for 1 h at RT followed by detection by chemiluminescence (SuperSignal Pico, Pierce). The following primary antibodies were used: anti-MCU (1:1000, Sigma-Aldrich HPA016480), anti-MICU1 (1:1000, Sigma-Aldrich HPA037480), anti-MICU2 (1:1000, Sigma-Aldrich HPA045511), anti-EMRE (1:1000, Santa Cruz sc-86337), anti-CLPB (1:1000, ProteinTech 15743-1-AP), anti-GRP75 (1:1000, Santa Cruz sc-133137), anti-LETM1 (1:1000, Abnova M03), anti-MITOK (1:10000, Sigma-Aldrich Cat #HPA010980), anti-OPA1 (1:1000, BD biosciences Cat #612606), anti-OXPHOS (1:1000, Abcam Cat #ab110413), anti-GAPDH (1:1000, Santa Cruz, Cat #sc-25778), anti-PDH (1:1000, Cell Signaling Technology Cat #2784), anti-AFG3L2 (1:1000, Thermo Fisher Scientific Cat #PA5-95347), anti-YME1L (1:1000, Sigma-Aldrich Cat #SAB1301252), anti-eIF2-α (1:1000, Cell Signaling Technology, Cat #5324), anti-phospho-eIF2-α (Ser51) (1:1000, Cell Signaling Technology, Cat #3597), anti-TMEM65 (1:1000, Thermo Fisher Scientific, Cat #PA5-112762, anti-Tom20 (1:1000, Cell Signaling Technology, Cat #42406).
HAP1 WT and HAP1 CLPB KO cells were washed two times with PBS and then harvested with 0.05% trypsin-EDTA (Gibco, #25300054). Immediately, the cells were lysed with RIPA buffer supplemented with protease inhibitors (leupeptin, antipain, pepstatin, 1 μg/ml of each) and PMSF 1 mM. Lysates were centrifuged at 12,000 rpm for 10 min at 4 °C to remove debris, aggregates, and insoluble proteins. The resulting supernatants, containing the extracted proteins and indicated as whole cell lysates were quantified using the DC protein assay kit (Bio-Rad, #5000113), and 25 μg of protein was loaded in a NuPAGE 10% and 4–12% Bis-Tris gels (Invitrogen, #NP0322BOX) and then separated electrophoretically in reducing and non-reducing conditions. After electrophoretic separation, proteins were transferred onto LF PVDF membranes (Bio-Rad, #1704275) and those were blocked for 1 h at room temperature, following the incubation overnight at 4 °C with the primary antibodies. The following antibodies were used: anti-CLPB (Proteintech, #15743-1-AP, 1:1000), anti-MCU (Sigma, #HPA016480, 1:500), anti-MICU1 (Sigma, #HPA037480, 1:500), anti-MICU2 (Abcam, #ab101465, 1:1000), anti-EMRE (Bethyl, #A300-BL19208, 1:1000), anti-TOM20 (Proteintech, #667771, 1: 1000), anti-HSP70 (Thermo Scientific, #MA3-028, 1:1000), and anti-OPA1 (BD Lab, #612606, 1:1000). After antibodies incubation, membranes were washed three times with TBS-Tween for 10 min and then incubated with fluorescence secondary antibodies (Licor, 1:5000) for 1 h in shaking at room temperature in the dark. Proteins were detected with chemiluminescence using the Azura Biosystems software. Proteins of interest as well as loading control bands were quantified by densitometry using ImageJ software (NIH, Bethesda, USA). Western blots shown throughout the paper are representative of at least three independent experiments.

Gastrocnemius muscle was homogenized in T-PER (Thermo Fisher Scientific) with a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail (Roche) that effectively lysed membrane and intercellular protein⁵⁸ . The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific). Briefly, 10 µg of protein sample per well was transferred to 7.5% polyacrylamide gels (Wako) and then transferred to a PVDF membrane by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Membranes were incubated overnight with the following primary antibodies: anti-AR (dilution 1:1000, Santa Cruz) and anti-ubiquitin (1:1000, Cell Signaling Technology)^{59 (link)}. The membranes were washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (GE Healthcare). Bands were detected using ECL Prime (GE Healthcare) and quantified using ImageJ software.
We also measured the whole protein simultaneously. Briefly, 10 µg of protein sample per well was transferred to 10% polyacrylamide gels (Wako) and stained with Coomassie Brilliant Blue (TaKaRa). Analysis was performed using ImageJ software.

Cells were washed three times with ice-cold PBS and then lysed in lysis buffer [composed of 20 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 400 mM NaCl, 0.5% NP-40, and 5% glycerol] containing 1/10 volume of Phosphatase Inhibitor Cocktail (Roche, Switzerland) and 1 mM dithiothreitol (Sigma-Aldrich, USA). Quantification of protein extracts was carried out using Quick Start Bradford Dye Regent (Bio-Rad, USA) and denatured at a temperature of 95°C for 10 min, separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to the polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Germany). Membrane were blocked in 5% bovine serum albumin (Sigma-Aldrich, USA) for 1 hour in room temperature and then incubated overnight, rotating at 4°C with the primary antibodies as following: ZDHHC2 (Santa Cruz Biotechnology, sc-515204), β-actin [Cell Signaling Technology (CST), 8457L], glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Proteintech, 60004-1-Ig), B-RAF (Santa Cruz Biotechnology, sc-5284), C-RAF (Santa Cruz Biotechnology, sc-7267), TBK1 (CST, 3013s), p-TBK1 (CST, 5483s), JNK (CST, 9252s), p-JNK (CST, 9251s), P65 (CST, 4764s), p-P65 (CST, 3033s), ERK1/2 (CST, 4695s), p-ERK1/2 (CST, 4370s), P38 (CST, 2307s), p-P38 (CST, 4511s), AKT (CST, 4691s), p-AKT (CST, 4060s), MEK1/2 (CST, 8727s), p-MEK1/2 (CST, 9154s), c-JUN (CST, 9165s), p-c-JUN (CST, 9164s), c-FOS (CST, 2250s), p-FOS (CST, 5348s), p-B-RAF (CST, 2696s), p-C-RAF (CST, 9421s), hemagglutinin (HA) tag (CST, 3724S), Myc-tag (Santa Cruz Biotechnology, sc-40), and DDDDK-Tag (Abclonal, AE092). The membranes were incubated with appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies [goat antimouse IgG (H + L), 31430; goat antirabbit IgG F(ab′)2, 31234, Thermo Fisher Scientific] at room temperature for 1 hour and detected with Plus-ECL (FDbio Science, China) according to the manufacturer’s protocol.