Bcl 2

Myocardial tissue and cell suspension was lysed by RIPA lysis buffer (MB-030-0050, Multi Sciences Biotech, Hangzhou, China). BCA Protein Assay Kit (23225, Thermo Fisher, USA) was adopted to determine protein concentrations. After equal quantities, total proteins were separated in SDS-PAGE gel (4-20%, ACE Biotechnology, Xiangtan, China), and the bands were transferred onto PVDF membranes (03010040001, Merck, USA). After blocked with 5% skimmed milk and washed with tris-buffered saline containing 0.1% tween-20 (TBST), protein bands were blotted with primary antibodies at 4° C overnight. After washing with TBST, protein bands were blotted with HRP conjugated secondary antibody and monitored using ECL buffer (32209, Thermo Fisher). Quantifications of western blotting were measured with Fusioncapt advance software. Antibody information: Bax (HUABIO, USA, ET1603-34, 1:1000), Bcl2 (HUABIO, ET1702-53, 1:1000), Cleaved-caspase3 (HUABIO, ET1608-64, 1:1000), IL-10 (Servicebio, Wuhan, China, GB11108, 1:1000), TLR4 (Servicebio, GB11519, 1:1000), α-SMA (Servicebio, GB111364, 1:1000), MMP-9 (Abcam, Cambridge, UK, ab76003, 1:1000), Collagen I (Abcam, ab34710, 1:1000), GAPDH (CST, USA, 5174S, 1:1000). HRP-conjugated secondary antibody (HUABIO, HA1001, and HUABIO, HA1006, 1:50000).

To investigate the associated protein activation, Western blotting analysis was performed. The protein samples were mixed with sample loading buffer (P0015F, Beyotime, China) and subjected to heat at 95 °C for 5 min before being separated on 10% SDS-PAGE gels, and subsequently transferred onto polyvinylidene fluoride membranes. The used antibodies were: FOXO1 (1:1000, 2880 s, Cell Signaling Technology, USA), VCAN (1:500, ET7107-09, Huabio, China), BAX (1:3000, ET1603-34, Huabio, China), BCL-2 (1:3000, ER0602, Huabio, China), neuronal nuclear antigen (NeuN; 1:1000, 36,662, Cell Signaling Technology, USA), MBP (1:1000, PA1-10,008, Thermo Scientific, USA). Finally, the blots were scanned and analyzed by ImageJ software. The normalized band intensities against the corresponding housekeeping protein β-actin (1:1000, 3700 s, Cell Signaling Technology, USA) were calculated for precise comparison.

Cells were harvested and lysed (0 °C, 30 min) in RIPA reagent (Beyotime, China), containing PMSF and a protease and phosphatase inhibitor cocktail (Roche, Switzerland). The protein concentration of each sample was measured by the BCA Protein Assay kit (Thermo, America). Equal amounts of proteins (30 µg each) were incubated at 95 °C for 10 min in an SDS buffer. Proteins were separated by 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore) after incubation overnight with primary antibodies against TLR4 (1:1000; Affinity, America), LC3B (1:1000; Abcam, America), p62 (1:1000; CST), ATG5 (1:1000; Sigma, Germany), p-p65 (1:1000; CST), p-p38 (1:1000; CST), BAX (1:500; CST), BCL-2 (1:500; HUABIO), Caspase-3 (1:1000; Proteintech, America), Caspase-8 P18 (Santa Cruz, America), and GAPDH (1:5000; Proteintech, America) at 4 °C. The membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000; Beyotime, China) and were then visualized using chemiluminescence (Thermo, America) and quantified by ImageJ software (National Institutes of Health; version 1.45).

After 48 h of HEA treatment, SGC-7901 cells were collected, total protein was extracted, and Western blot analysis was performed as previously described [13 (link)]. Primary rabbit antibodies against β-actin (1:3000), AIF (1:2000), Bcl-2 (1:2000), and Bax (1:2000) were purchased from HUABIO (Hangzhou, China). Primary antibodies against caspase-3 (1:2000), caspase-8 (1:2000), caspase-9 (1:2000), caspase-12 (1:1000), cytochrome C (1:500), poly(ADP-ribose) polymerase (PARP) (1:2000), cleaved-PARP (1:2000), and p53 (1:1000), were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against ATF4 (1:1000), CHOP (1:500), p62 (1:1000), and Beclin1 (1:1000) were obtained from Proteintech (Rosemont, IL, USA). Primary antibodies against LC3 (1:500), ATG5 (1:1000), and ATG12 (1:1000) were obtained from MBL (Nagoya, Japan). Anti-rabbit lgG horse-radish peroxidase-conjugated secondary antibody (1:3000) was purchased from Abcam (Cambridge, MA, USA).

Murine ovarian tissue and drug-treated KGN cells were subjected to protein extraction in RIPA lysis and extraction buffer (Thermo, USA). Protein concentrations were quantified using the BCA protein assay kit (Thermo, USA). The protein samples were fractionated by SDS‒PAGE and then transferred to PVDF membranes (Millipore, USA). Subsequently, the membrane was blocked using 5% skim milk for 1 h. Then, the sections were incubated overnight at 4 °C with the corresponding primary antibodies, including pATM, PGC-1α, TERF2 (ABclonal, China), SIRT1, p53 (Proteintech Group, China), Bax, Bcl-2, and β-actin (HuaBio, China), and then probed with secondary antibodies. Protein chemiluminescence was detected using the ECL chemiluminescence reagent (Thermo, USA). Finally, exposure was performed using an exposure device.

Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China, P0013B). Total protein was quantified by BCA protein assay (Bio‐Rad, Berkery). Each sample was adjusted to equal amount of protein using 5X loading buffer for loading. The samples were separated by SDS‐PAGE, transferred to a polyvinylidene fluoride membrane, and immunoblotted with the following antibodies: GDF11 (DGDF80, R&D Systems,Emeryville, CA,USA), HGF (ab83760, Abcam, USA), VEGFR1 (ET1605‐11,Huabio，China), CD31 (#77699, Cell Signalling TechnologyBoston,MA, USA), VEGFR2 (#9698, following Abs are all from Cell Signalling Technology,USA), phospho‐p44/42 (Thr202/Try204 phospho‐ERK1/2,#4370), p44/42 MAPK (Erk1/2,#4696), BCL2 (#2827), BAX (#14796), Cleaved Caspase3 (#9661), phospho‐Smad2 (Ser465/Ser467,#18338), phospho‐Smad3 (Ser423/425, # 9520), Smad2(#5339), Smad3(#9523), anti‐β‐actin (#3700), EIF4E (R1512‐8,Huabio, China), Phospho‐eIF4E (S209) (ET1608‐66,Huabio, China) and VEGF ( ER30607,Huabio, China),at 4°C overnight. After incubation of the membranes with peroxidase‐conjugated secondary antibodies (Cell Signalling Technology,USA), bands were visualized using enhanced chemiluminescence reagents (Bio‐Rad,Los Angeles, CA,USA).