Bax 50599 2 ig

RIPA lysis buffer (#P0013B; Beyotime) was used to extract total protein from cells according to the manufacturer's protocol. Protein was quantified using a BCA protein determination kit (#23225, Thermo Fisher Scientific, USA). Following the addition of loading buffer to the protein samples, the mixture was kept in water at 100°C for 5 min and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed. Protein samples were then transferred to a polyvinylidene fluoride membrane. This membrane was sealed with 5% skim milk and blocked for 2 h at room temperature. Subsequently, it was incubated overnight at 4°C with the following primary antibodies: Bcl-2 (3498; CST), Bax (50599-2-Ig; Proteintech), cleaved caspase-3 (9664; CST), CDK2 (2546; CST), and GAPDH (60004-1-Ig; Proteintech). Peroxidase-AffiniPure goat anti-rabbit IgG (H + L; 111-035-003; Jackson) and peroxidase-AffiniPure goat anti-mouse IgG (H + L; 115-035-003; Jackson) were used as secondary antibodies. For enhanced chemiluminescence, Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was used to detect the proteins, with GAPDH as the internal reference.

Total proteins were collected from target cells with a Cell Mitochondria Isolation Kit (Beyotime, Shanghai, China). The samples were incubated at 4 °C for one night with the primary antibodies METTL3 (15073-1-AP, Proteintech), Bcl-2 (ab196495, Abcam), Bax (50599-2-Ig, Proteintech), Caspase 9 (10380-1-AP, Proteintech), Caspase 3 (19677-1-AP, Proteintech), cleaved PARP-1 (ab32064, Abcam), E-cadherin (BF0219, Affinity), N-cadherin (AF4039, Affinity), vimentin (10366-1-AP, Proteintech), PTEN (22034-1-AP, Proteintech), YTHDF2 (ab220163, Abcam), p53 (CSB-PA07889A0Rb, CUSABIO Life Sciences, College Park, USA), c-Myc (10057-1-AP, Proteintech), NF-κb (ab76302, Abcam), Zeb1 (ab180905, Abcam), Zeb2 (ab191364, Abcam), Snail (ab180714, Abcam), Twist1 (ab50887, Abcam); after that, the samples were incubated at room temperature for 1 h with goat anti-rabbit IgG polyclonal antibody (Abcam) or goat anti-mouse IgG polyclonal antibody (Abcam). GAPDH levels were used as an endogenous control for visualization employing the enhanced chemiluminescence (ECL) detection method (Thermo Fisher Scientific).

Equal amounts of protein extracted from cardiac muscle tissues with the use of a buffer were separated on SDS-polyacrylamide gels and were electrophoretically transferred to polyvinylidene fluoride membranes. After blocking the membranes with 5% nonfat milk, they were incubated overnight at 4 °C with primary antibodies for PTEN (ab31392; 1:500) and p-PTEN (ab131107; 1:500) purchased from Abcam (Cambridge, UK), Akt (2920; 1:2000); p-Akt (4060S; 1:2000), mTOR (2983; 1:1000), and p-mTOR (2971S; 1:1000) purchased from Cell Signaling Technology (Danvers, MA, USA); GAPDH (1:1000) purchased from Beyotime (Jiangsu, China); Bcl-2 (AF6139; 1:1000) and BNP (DF6902; 1:500) purchased from Affinity Biosciences (Cambridge, UK); and Bax (50599-2-Ig; 1:1000) purchased from Proteintech Group (Chicago, IL, USA). They were then incubated with HRP-conjugated secondary antibody and the immunoreactive bands were subsequently detected with a chemiluminescence kit.

Cell culture materials were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against p-STAT3 (Tyr705) (#9145, 1:1000), STAT3 (#30835, 1:2000), p-p65(Ser536) (#3033, 1:1000), p65 (#8242, 1:2000), and α-SMA (#19245, 1:2000) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against IL-1β (sc-12742, 1:2000), CD34 (sc-7324, 1:2000), VCAM-1 (sc-13160, 1:2000), and EPAS-1 (sc-13596, 1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against CD68 (28058-1-AP), ICAM-1 (60299-1-Ig, 1:2000), MCP-1 (66272-1-Ig, 1:2000), Bax (50599-2-Ig, 1:2000), Bcl-2 (12789-1-AP, 1:2000), cleaved-caspase-3 (19677-1-AP, 1:2000), GAPDH (10494-1-AP, 1:2000), FN1 (15613-1-AP, 1:2000), and SOX9 (67439-1-Ig, 1:2000) as well as secondary antibodies (Goat anti-mouse, SA00001-1; Goat anti-rabbit, SA00001-2) were purchased from Proteintech Group (Chicago, IL, USA). Recombinant human interleukin-1 receptor antagonist (IL-1Ra) (SRP3084) was obtained from Sigma (St. Louis, MO, USA), Stattic (inhibitor of STAT3) and Colivelin, (activator of STAT3) were purchased from MedChemExpress (Shanghai, China). Unless otherwise indicated, the remaining reagents used in this study were obtained from Sigma.

All cells were lysed in radioimmunoprecipitation assay (RIPA) buffer plus 1% phosphatase inhibitors (New Cell & Molecular Biotech, Suzhou, China). All lysates were quantitated with a BCA Assay and mixed with 5× SDS loading buffer and boiled for 10 min. Equal amounts of total protein were loaded into each lane of a 10% polyacrylamide gel and separated by electrophoresis, followed by transfer onto a polyvinylidene fluoride (PVDF) membrane (Invitrogen). Membranes were blocked in 5% nonfat dry milk/Tris-buffered saline-0.5% Tween 20 and then incubated with primary antibodies overnight at 4 °C. GAPDH was used as a loading control. The membranes were further probed with horseradish-peroxidase-conjugated secondary antibody for 1 h at room temperature. The protein levels were detected by using the enhanced chemiluminescence (ECL) Immunoblot Analysis Detection System.
The primary antibodies used were as following: YAP (ab52771, Abcam, Cambridge, UK); phospho-YAP (ser 127) (ab76252, Abcam); ERK1/2 (#4695, CST, Danvers, MA, USA); phospho-ERK1/2 (#4370, CST); p38 MAPK (#8690, CST); phospho-p38 MAPK (#4511, CST); BAX (50599-2-Ig, Proteintech, Rosemont, USA); BCL2 (12789-1-AP, Proteintech); and VEGF (19003-1-AP, Proteintech).

Cells were treated the same as 4.5. and then washed twice with ice-cold PBS and were lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China) after the treatment with metformin or/and EGCG co-cultured with MG. The Supernatant was collected, and concentration was determined by BCA protein assay kit (Solarbio, Beijing, China). The same amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% skim milk for 2 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies Bcl-2 (12789-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), Caspase-3 (A11040, ABclonal), Cytochrome C (A4912, ABclonal), GSK-3β (A2081, ABclonal), BACE-1 (AF6273, Beyotime), APP (A17911, ABclonal), and β-actin (AC038, ABclonal). The membranes were washed with 1% TBST three times, and then incubated with secondary antibodies (1:3000 dilution) for 1 h at room temperature. Finally, the bands were visualized using an ECL kit (Tanon, Shanghai, China).

AFCs were lysed with RIPA lysis buffer (P0013B, Beyotime), and the protein concentration was determined by BCA assay kit (PC0020, Solarbio); 1/5 volume of loading buffer was added to the lysate, and then the sample was placed in boiling water for 10 min. Proteins are separated by sulfate–polyacrylamide gel electrophoresis (SDS‒PAGE) techniques and transferred to the PVDF membrane. PVDF membrane was blocked by 5% skim milk for 1 h, and then the membrane was incubated with primary antibodies at 4° overnight. Primary antibodies contain calpain1 (10,538–1-AP, Proteintech), calpain2 (11,472–1-AP, Proteintech), Bax (50,599–2-Ig, Proteintech), cleaved-Caspase 3 (9664, Cell Signaling Technology, MA, USA), Piezo1 (5939–1-AP, Proteintech), and Gapdh (60,004–1-Ig, Proteintech). PVDF membrane was washed by TBST and then was incubated with the secondary antibody for 1 h. The secondary antibodies contain Goat Anti-Mouse IgG (H + L) HRP (abs20001, Absin, Shanghai, China) or Goat Anti-Rabbit IgG (H + L) HRP (abs20002, Absin). The expression of the target protein was detected by Clarity Western ECL Subs (1705060SP, Bio-Rad, Universal Hood III, CA, USA) and ChemiDoc imaging system (Bio-Rad) and analyzed by ImageJ software. All results were quantified and normalized to GAPDH.