Mitochondrial protein extraction kit

The procedure of western blot was described in detail in our previous publication [41 (link)]. The total protein samples from Sf9 cells were extracted by CytoBuster^TM Protein Extraction Reagent (Novagen, Kenilworth, NJ, USA), and the cytoplasmic and the mitochondrial proteins were extracted by Nuclear and Cytoplasmic Protein Extraction Kit and Mitochondrial Protein Extraction Kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s protocol. The Bradford method was used to detect protein concentration. In brief, 20 μg of protein from different samples was subjected to 12% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). The membranes were blocked in tris-buffered saline (TBS) supplemented with 5% fat-free milk at 4 °C overnight and incubated with the primary antibodies anti-Sf-IBM1 and anti-GAPDH (Beyotime Biotechnology, Shanghai, China) by the dilution ratio of 1:3000 at room temperature for 2 h. Then, membranes were washed with TBS buffer supplemented with 0.05% Tween 20 (TBST) and incubated with the diluted horseradish peroxidase-conjugated secondary antibodies at room temperature for more than 2 h. The protein bands were visible by enhanced chemiluminescence (ECL) western blot detection reagents (Bio-Rad, Hercules, CA, USA) and recorded by ChemiDoc^TM MP imaging system (Bio-Rad).

Sf9 cells treated with 7.5 μg/mL ZC-14 for 6, 12, 18 and 24 h were harvested by centrifugation (600× g, 5 min) and washed twice with PBS. Total protein was extracted by CytoBuster^TM Protein Extraction Reagent (#71009, Novagen, Kenilworth, NJ, USA) and incubated at 25 °C for 10 min, then centrifuged with 13,000× g at 4 °C for 10 min. The supernatants were used as the total proteins samples for western blot. The mitochondrial and cytosolic proteins were isolated by mitochondrial protein extraction kit (#KGP850, KeyGEN, Nanjing, China) according to manufacturer’s protocol. Bradford method was used to determine the protein concentration.
Identical quantities of protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to 0.45 μm polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk at room temperature for 2 h and then incubated with primary antibodies diluted 1:2000 in tris-buffered saline (TBS; 100 mM Tris-HCl, pH 7.5, 0.9% NaCl) overnight at 4 °C, followed by secondary antibody conjugated with horseradish peroxidase (diluted 1:4000 in TBS) for 2 h. The protein label was visualized by ECL detection system (Bio-Rad, West Berkeley, CA, USA).