Anti bcl 2

Protein was extracted from glioma cells and measured through the BCA kit (Beyotime Biotechnology, China) [18 (link)]. Then, the protein was extracted using SDS-PAGE (10%) and transformed into PVDF membranes (Millipore, USA). Afterwards, membranes were incubated using 5% skimmed milk and incubated with primary antibodies under 4°C overnight. The antibodies are as follows: anti-Bax (1: 2, 000, bs-28034R, Bioss, China), anti-Bcl-2 (1: 2, 000, bs-4563R, Bioss, China), anti-MMP-2 (1: 2, 000, bs-20705R, Bioss, China), anti-MMP-9 (1: 2, 000, bs-22502R, Bioss, China), anti-Cleaved caspase-3 (1: 2, 000, bsm-33199M, Bioss, China), anti-Cleaved caspase-9 (1: 2, 000, bs-3082R, Bioss, China), anti-Cox-2 (1: 2, 000, bs-10411R, Bioss, China), and anti-β-actin (1: 2, 000, bs-0061R, Bioss, China) with β-actin being the endogenous control. Afterwards, membranes were incubated for 1 h using a secondary antibody (1: 2, 000, bs-0311P-HRP, Bioss). Finally, ECL (Millipore, USA) was utilized to observe protein blots and quantified using ImageJ software (version 4.3; National Institutes of Health).

Spleen samples were lysed in RIPA buffer containing Halt protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, San Diego, CA, USA) and incubated for 45 min at 4 °C. Samples of lysate equivalent to 20 μg of protein were separated by 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and proteins were transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with Bovine Serum Albumin (Gibco, Carlsbad, CA, USA) and incubated with the following primary antibodies; anti-MCL1 (Proteintech, Chicago, IL, USA), anti-BCL2 (Bioss Biotechnology, Beijing, China), anti-BAX (Bioss Biotechnology), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam, Cambridge, UK) for 15 h at 4 °C. The membranes were washed again and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG (both Thermo Fisher Scientific, San Diego, CA, USA), or mouse anti-goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA). Bound proteins were visualized using SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, San Diego, CA, USA), and the membranes were analyzed using an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).

The heart tissues and H9 c2 cells in each group were added with RIPA Lysis Buffer (Beyotime) and homogenised on ice. After centrifugation, the supernatant was taken and denatured by protein-loading buffer. After being electrophoresed, the protein was transferred to the nitrocellulose membrane (IB24001, Thermo Fisher Scientific, Shanghai, China), then blocked with skim milk powder, and incubated with primary antibodies (anti-SOD1 (Bioss), anti-SOD2 (Bioss), anti-Calcipressin 1/DSCR 1 (Bioss), anti-RhoA (Bioss), anti-Calpain 2 (Bioss), anti-Caspase-3 (Bioss), anti-phospho-NFKB p65 (Ser281) (Bioss), and anti-Bcl-2 (Bioss)) overnight. Next day, the corresponding secondary antibody was used for continuous incubation, the images were collected and processed after exposure to enhanced chemiluminescence (ECL), and gapdh/β-actinwas used as an internal control. The protein expression level was analysed with ECL-plus reagent (GE Healthcare, Shanghai, China).

Berberine (purity: ≥98%) and cisplatin (purity: ≥99%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-MMP-2, anti-MMP-9, anti-Bcl-2, anti-Bax, anti-CyclinD1, anti-CDK4, anti-JNK, anti-phospho-JNK, anti-ERK, anti-phospho-ERK, anti-P38, and anti-phospho-P38 primary antibodies were purchased from Bioss (Beijing, China). Goat antimouse IgG and goat antirabbit IgG secondary antibodies were purchased from Life Science (Santa Cruz, CA, USA).

Total protein was extracted from the tissue in RIPA lysis buffer (containing protease and phosphatase inhibitor mixtures) by using a tissue homogenizer, followed by clearing tissue debris by centrifugation at 13000 rpm at 4°C for 20 min. Fifty micrograms of protein was loaded per lane and separated by 10% SDS-PAGE gel electrophoresis and then transferred onto PVDF membranes. Blocking was carried out in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 1 h at room temperature. The membranes were incubated with primary antibody rabbit anti-Bax (diluted 1 : 300, Bioss, Beijing, China); anti-Bcl-2 (diluted 1 : 300, Bioss, Beijing, China), overnight at 4°C and with secondary antibody (1 : 40000 dilution of goat anti-rabbit) conjugated to horseradish peroxidase (Boster, Wuhan, China) for 1 h at room temperature on the following day. Immunoblotting signal was detected by ECL (enhanced chemiluminescence) on chemiluminescent films following exposure to an X-ray. For densitometric analyses, the blots were scanned and quantified using BandScan software (Glyko Biomedical, Canada), and the result was expressed as the ratio of target gene immunoreactivity to GAPDH immunoreactivity.