Anti tom20

The antibodies were as follows: anti-Rab32 (Proteintech, #10999-1-AP, 1:1000), anti-MMP2 (Proteintech, #10373-2-AP, 1:1000), anti-MMP9 (Proteintech, #10375-2-AP, 1:1000), anti-β-Actin (CST, #4970, 1:1000), anti-N-cadherin (CST, #13116, 1:1000), anti-E-cadherin (CST, #3195, 1:800), anti-Vimentin (CST, #5741, 1:1000), anti-p-Drp1(Ser616) (CST, #3455, 1:1000), anti-p-Drp1(Ser637) (Abmart, #TD2980, 1:1000), anti-Drp1(CST, #8570, 1:1000), anti-Tom20 (Abcam, #ab56783, 1:1000), anti-p-ERK_1/2 (CST, #4370, 1:2000), anti-ERK_1/2 (CST, #4695, 1:2000). All secondary antibodies (HRP-conjugated anti-rabbit and anti-mouse, #A0208 and #A0216, 1:2000) were purchased from Beyotime Biotechnology (Shanghai, China). Anti-HA-tag (CST, #3724, 1:50 for IP and 1:1000 for western blot) and anti-IgG antibody (Servicebio, #GB23301, 1:100 for IP). Mdivi-1 (#S-7162), ERK_1/2 inhibitor SCH772984 (#S-7101) were purchased from Selleck.

The Lowry method was used to evaluate the protein content after the cells had been seeded in 6-well plates, cultured in complete media, and lysed using ice-cold lysis buffer supplemented with protease inhibitor cocktails (Roche Molecular Biochemicals, Mannheim, Germany) (Biorad DC Protein Assay, MA, USA). In the running buffer, 25 µg of protein from each sample was placed onto a polyacrylamide gel and electrophoretically separated. The proteins were blotted onto a Hybond-P PVDF membrane following electrophoresis (Amersham Biosciences, Buckinghamshire, UK). The membrane was exposed to anti-TOM20 (mouse, 1:1000; AbCam, Cambridge, UK), anti-VDAC1 (mouse, 1:1000; AbCam, Cambridge, UK), and anti-BNIP3 (rabbit, 1:1000; AbCam, Cambridge, UK) after blocking with a 10% skim milk solution. Following washing, the membrane was incubated with HRP-conjugated anti-rabbit secondary antibody (1:3500; PerkinElmer, MA, SUA) or anti-mouse secondary antibody (1:10000; PerkinElmer, MA, USA). According to the manufacturer’s instructions, the signal was seen using an improved chemiluminescent kit from Amersham Biosciences, and then it was examined using a Molecular Imager VersaDoc MP 4000 (Biorad, Hercules, CA, USA). Proteins were normalized to β-ACTIN (mouse, 1:7000, AbCam, Cambridge, UK) and GAPDH (rabbit, 1:2000, Cell Signaling, Danvers, MA, USA).

Cells or tissue samples were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors (Calbiochem). The protein concentration was determined with a BCA Assay Kit (CWBIO). Equal amounts of protein were subjected to 12% SDS‐PAGE and then transferred to PVDF membranes (Merck Millipore). The PVDF membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies overnight at 4°C: anti‐IL‐1β (Abcam), anti‐ICAM‐1 (Proteintech), anti‐HO‐1 (ABclonal), anti‐NrF2 (Abcam), anti‐p‐NFκB (Cell Signaling Technology (CST)), anti‐NFκB (CST), anti‐TFAM (ABclonal), anti‐TOM20 (Abcam), anti‐acetylated‐SOD2 (Ac‐SOD2, Abcam) and anti‐SOD2 (Proteintech). After washing with PBST, the PVDF membranes were incubated with HRP‐conjugated secondary antibody (1:2000, ABclonal, USA) at 37°C for 1 h. Protein bands were detected with a chemiluminescence kit (Merck Millipore) and quantified by ImageJ software (NIH).

Heart tissues or cells were collected and lysed with RIPA buffer (Beyotime, P0013C) containing protease inhibitors (Selleck, B15002). The samples (20 µl) were used to determine the total protein concentrations with BCA reagent kit (Applygen, P1511-1). Protein samples were heat-denatured for 5 min at 95°C with the sample loading buffer, and 20 µg of total protein per sample were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, IPVH00010). The membranes were blocked with 5% BSA for 30 min and incubated overnight at 4°C shaking with primary antibodies. The membranes were then washed in Tris-buffered saline (TBS, pH 7.6) with 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies (1:4000, ZSGB-BIO, ZB2301) for 2 h at room temperature. Bands were visualized using ECL Detection Reagent (Applygen, P1050), and quantify analysis was performed using Gel-Pro Analyzer System. The primary antibodies used were presented as follow: anti-LC3B (1:5000, Sigma, L7543); anti-p62/SQSTM1 (1:5000, Sigma, P0067); anti-PINK1 (1:1000, Abcam, ab23707); anti-TOM20 (1:1000, Abcam, ab78547); anti-α-Actin (1:5000, Abcam, ab156302).

Antibodies used include anti-Flag M2 (Sigma, F3165), anti-TOM20 (Abcam, ab78547), anti-BAG5 (Cusabio, CSB-PA890743HA01HU), anti-Actin (Sigma, A2066), anti-GAPDH (Cell Signaling Technology, 14C10), anti-GFP (Abcam, Ab290), anti-Parkin (Cell Signaling Technology, 2132S), anti-Caspase-3 (Enzo, ADI-AAP-113-D), anti-Cleaved Caspase-3 (Cell Signaling Technology, 9664S), anti-Tubulin (Cell Signaling Technology, 2148S), anti-Mcl-1 (Cell Signaling Technology, 4572S), anti-HA-tag antibody conjugated sepharose beads (Cell Signaling Technology 3956), and anti-HA (Sigma, 11867423001).

Western blotting was carried out as described before.^{22 (link)} Total protein was extracted by RIPA buffer containing protease cocktail inhibitor. All protein samples were quantified. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk in TBS-T (BD Pharmingen), PVDF membranes were incubated with the primary antibody overnight at 4 °C and then with the secondary antibody for 1 h at room temperature. The following antibodies were used: anti-MFN1 (1:1000, Abcam), anti-MFN2 (1:1000, Abcam), anti-Opa1 (1:1000, Abcam), anti-DNM1L (1:1000, Abcam), anti-MFF (1:1000, Abcam), anti-TOM20 (1:1000, Abcam), anti-E-cadherin (1:1000, Abcam), anti-N-cadherin (1:1000, Abcam), anti-Snail (1:1000, Abcam), anti-Twist (1:1000, Abclonal), anti-Zeb1 (1:1000, Abclonal), anti-Slug (1:1000, Abclonal) anti-Vimentin (1:1000, Abcam) and anti-β-actin (1:1000, Abcam). Protein bands were detected by using image acquisition using ImageQuant™ LAS 4000 (GE Healthcare Life Sciences).

Western blot analysis was performed as previously described^{52 (link)}. Briefly, the cardiomyocytes were resuspended in lysis buffer (20 mM Tris pH 7.5, 2 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol, 250 mM sucrose, 0.1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100) with a protease inhibitor cocktail at 4 °C for 1 h. The samples were centrifuged at 13,000 rpm at 4 °C for 30 min. Subsequently, 30 µg of the supernatants was boiled, separated on 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Then, the membranes were incubated at 4 °C overnight with the following diluted primary antibodies. The anti-ND1, anti-ND2, anti-ND3, anti-ND4, anti-ND5, anti-COX1, anti-Cytb, anti-Tom20, and anti-ND6 antibodies were from Abcam. The anti-COX3 and ATP6 antibodies were from Abclonal. The anti-COX2 antibody was purchased from Bimake. The anti-ATP8 and anti-GAPDH antibodies were purchased from Santa Cruz. The anti-ND4L antibody was purchased from Invitrogen. After washing four times with 1× PBST, the membrane was incubated with the appropriate secondary antibody conjugated with horseradish peroxidase (HRP). Antigen–antibody complexes were visualized by enhanced chemiluminescence. The densitometric measurements were analyzed by using Image J software.