Anti bcl 2

The indicated cells (4–5 × 10⁵/well) were seeded in 6-well plates and cultured for 24 h, then cells treated with or without 4 μM KA. Subsequently, cells were lysed in RIPA buffer containing protease inhibitors. Supernatants were collected and subjected to 10% SDS-PAGE, and transferred onto PVDF membranes. The membranes were then incubated overnight with primary antibodies. The following antibodies were used: Anti-E-cadherin (Cat#:76055, Abcam Biotechnology), Anti-Vimentin (Cat#:8978, Abcam Biotechnology), Anti-t-ERK (Cat#:4695, Cell Signaling Technology), Anti-p-ERK (Cat#:4370, Cell Signaling Technology), Anti-Caspase-3 (Cat#:9662, Cell Signaling Technology), Anti-Bcl-2(Cat#: WL01556, Wanleibio, China), Anti-Bax (Cat#: 50599-2-Ig, Proteintech), Anti-β-Actin (Cat#: BS6007M, Bioworld Technology), and Anti-Ki-67(Cat#:550609, BD Pharmingen).

Granulosa cells were incubated with 3-NPA at 5.0 mmol/l for 24 h and then were harvested. The total protein was extracted using RIPA Lysis Buffer and 10% phenylmethanesulfonyl fluoride (Beyotime, China). Protein concentration was determined by using a BCA Protein Assay Kit (Beyotime, China). Approximately, the same amount of protein was separated using 10–12% SDS-PAGE, transferred electrophoretically onto the polyvinylidene membrane (Bio-Rad) and blocked with 5% nonfat dry milk. The primary antibodies used in the present study were anti-Caspase 3 (CST, U.S.A.), anti-Bcl-2 (Wanlei Biotechnology, China), anti-p53 (Wanlei Biotechnology, China), anti-Bax (Bioss Biotechnology, China), anti-β-actin (TransGen Biotechnology, China). The membrane was incubated with the primary antibody solution overnight at 4°C, and then washed four times with the TBST (TBS, 0.1% Tween 20). The corresponding secondary antibody (1:5000) was added and incubated at room temperature for 1 h. The protein bands were visualized by using the BeyoECL Plus (a chemiluminescence reaction; Beyotime, China) in an Image Lab software (Bio-Rad, U.S.A.). The bands were quantified using an ImageJ software (NIH, U.S.A.).

H9C2 cells with different treatment were lysed, quantified and subjected to SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes (Cat # 1,620,177, Bio-Rad, Hercules, CA, USA) which were incubated with 5% nonfat milk in TBST and followed by incubation of primary antibodies at 4°C overnight. Then the PVDF membranes were washed with TBST and incubated with secondary antibodies. Protein expression was detected using BeyoECL Moon (Cat # P0018FS, Beyotime) under a Tanon 5200 machine (Shanghai, China). The detailed primary and secondary antibodies were listed as below: anti-cleaved caspase 3 (Cat # E83-77, Abcam); anti-caspase 3 (Cat # ab32351, Abcam); anti-Bax (Cat # WL01637, Wanleibio, Shenyang, China); anti-Bcl-2 (Cat # WL01556, Wanleibio); anti-β-actin (Cat # WL01372, Wanleibio); anti-Nrf2 (Cat # WL02135, Wanleibio); anti-Sirt2 (Cat # ab211033, Abcam); anti-HO-1 (Cat # EP1391Y, Abcam); anti-GST (Cat # ab138491, Abcam); anti-NQO1 (Cat # 11,451-1-AP, Proteintech, Wuhan, China); anti-FOXO3a (Cat # 66,428-1-Ig, Proteintech); anti-GCLM (Cat # 14,241-1-AP, Proteintech); anti-Keap1 (Cat # 10,503-2-AP, Proteintech). IgG(H + L)(HRP-labeled Goat Anti-Rabbit IgG(H + L)) and IgG(H + L)(HRP-labeled Goat Anti-Mouse IgG(H + L)) were purchased from Beyotime (Beijing, China). All protein expressions were normalized by β-actin expression.

Total proteins of bovine myoblasts were prepared with radio immunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China). Proteins were fractionated on 12% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). Blots were incubated overnight with primary antibodies specific for anti-CyclinD1 (#ab226977), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #ab9485), anti-MyoD (#ab16148) (Abcam, Cambridge, UK), anti-INSR (#WL02857), anti-PCNA (#WL01804), anti-CDK2 (#WL01543), anti-CyclinE (#WL01072), anti-Bcl-2 (#WL01556), anti-P53 (#WL01919), anti-P21 (#WL0362), anti-MyoG (#WL01132), anti-MyHC (#WL02785), and anti-Bax (#WL01637) (Wanleibio, Haerbin, China) at 4°C. After incubation with secondary antibodies, the membranes were quantified with the ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).

Total proteins were collected using lysis buffer supplemented with protease inhibitors and phosphatase inhibitors. The protein concentration was determined using the bicinchoninic acid assay kit (Solarbio, Beijing) according to the manufacturer's protocol. Anti-GAPDH was obtained from Protein Tech. The primary antibodies of p21, p16, p53, BAX, MST2, p-LATS, YAP, p-YAP, RUNX2, ALP, ERK, p-ERK, AKT, p-AKT, and COL1 were purchased from CST (Danvers, MA). Anti-BCL-2 was purchased from Wanlei Biotech (Shenyang, China).

The HepG2, Hep3B, H22, and 293T cell lines were given as gifts by Professor Aiguo Wang (Dalian Medical University, Dalian, China), who purchased them from the Chinese Academy of Sciences, Beijing, China. The cells were grown in McCoy’s medium (Sangon Biotech, Shanghai, China), RPMI-1640, or Dulbecco’s modified Eagle’s medium (DMEM) (Sangon Biotech, Shanghai, China) with 10% fetal bovine serum (FBS) (CellMax, AusGeneX, Queensland, Australia). Human SPINK1-overexpressing (OE) and SPINK1 short hairpin RNA (shRNA) lentiviral vectors were generated by Applied Biological Materials (ABM, Nanjing, China), and the Transwell system was purchased from Nest (Southborough, MA, USA). The following commercially available antibodies were used: anti-SPINK1 (Abnova, Taipei, Taiwan) and anti-Bcl-2, anti-Bcl-XL, anti-Bax, anti-Bad, and anti-caspase-3 (all from Wanlei Biotech, Shenyang, China). The main chemicals or reagents used in this study were as follows: 5-fluoruracil (5-FU) (Shanghai Pharmaceutical Company, Shanghai, China), Lipo2000 (Invitrogen, Carlsbad, CA, USA), Cell Counting Kit-8 (CCK-8) (Xian Baiying Biotechnology Inc., Xian, China), puromycin (Solarbio, Beijing, China), and polybrene (Maokang Biotech, Shanghai, China).

Shengqi fuzheng injection (Z19990065) was provided by livzon Pharmaceutics ltd. (Zhuhai, China). For cell culture, SFI was dissolved in DMEM to different concentration gradients. Gefitinib was purchased from Aladdin Industrial Corporation (Shanghai, China). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Biosharp (Shanghai, China). Dulbecco's modified eagle medium (DMEM), DMSO, Penicillin- Streptomycin Solution, Annexin V-FITC kit were purchased from KeyGen (KeyGen, Nanjing, China). DAPI staining solution, EGF, BCA protein assay kit, Crystal Violet Staining Solution were purchased from Beyotime Biotechnology (Shanghai, China). Anti-p- EGFR(Tyr 1172), anti-EGFR, anti-MEK1/2, anti-p- ERK1/2(thr202/tyr204), anti-ERK1/2, anti-β-actin, goat anti-rabbit IgG H&L (HRP), anti-cleaved-caspase 3, anti-cleaved-caspase 9, anti-Bcl-2, anti-Bax antibodies were purchased from Wanlei Bio. (Shenyang, China). Anti-p-MEK1/2 (Ser217/221) antibody was purchased from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA).