Glyceraldehyde 3 phosphate dehydrogenase gapdh

Phloretin (PHL, PubChem CID: 24278652) and resveratrol (RES, PubChem CID: 445154), with a purity of 98%, was purchased from Aladdin (Shanghai, China). PHL and RES was dissolved in 0.5% sodium carboxyl methyl cellulose (CMC-Na) for in vivo experiments and in dimethyl sulfoxide (DMSO) for in vitro experiments [52 (link),53 (link)]. Glucose and streptozotocin (STZ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Haematoxylin-eosin (H&E) was purchased from Beyotime (Nantong, China). Masson’s trichrome kits were obtained from Solarbio (Beijing, China). The assay kits for glutamic oxalacetic transaminase (GOT) and creatine kinase (CK-MB) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies for TGF-β, collagen-1A1 (COL-1A1), IκB, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and secondary antibodies were purchased from Cell Signaling Technology (Danvers, USA). ANP and F4/80 were bought from Santa Cruz Technology (Santa Cruz, USA). RIPA lysis buffer was acquired from Boster Biological Technology (Wuhan, China).

Primary antibodies against total-p65, phospho-p65 (S536), total-c-Jun, phospho-c-Jun (Ser⁶³/Ser⁷³), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technologies (Danvers, MA, U.S.A.). Antibodies against NMBR weres purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Antibodies against α-smooth muscle actin (α-SMA) were purchased from Abcam (Cambridge, U.K.). Horseradish peroxidase (HRP)–conjugated anti-rabbit or anti-mouse secondary antibodies were purchased from Cell Signaling Technologies (Danvers, MA, U.S.A.). An ECL-plus kit was purchased from Advansta (MenloPark, CA, U.S.A.). NMB was purchased from Bachem (Bubendorf, Switzerland). The human IL-6 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Enzo Life Sciences (New York, NY, U.S.A.). Polybrene was purchased from Invitrogen (San Diego, CA, U.S.A.).

For western blotting, proteins from each sample were separated by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Cell Signaling Technology, MA, USA) at 300 mA for 100–120 min. The membrane was initially blocked in 5% bovine serum albumin for 2 h at room temperature and then incubated overnight at 4°C with primary heat shock protein- (HSP-) 70 (1 : 2000, Abcam, UK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1 : 1000, Cell Signaling Technology, MA, USA), histone deacetylase 5 (HDAC5) (1 : 1000, CST, USA), or integrin alpha-5 (ITGA5) antibody (1 : 1000, Cell Signaling Technology, MA, USA), respectively, followed by three or four washes with Tris-buffered saline, 0.1% Tween 20. The membrane was then incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 3000, Beyotime, China) for 2 h at room temperature. Protein expression was then observed by chemiluminescence using an Alpha Imager scanner (Tecan, Thermo Fisher Scientific, USA).

Protein was extracted from BMMs by RIPA cell lysis buffer (Cell signaling Technology, Danvers, MA, USA). Total protein (30 μg) was loaded on 10% Tris-HCl gels, electro-transferred to nitrocellulose membranes, blocked, and incubated overnight at 4 °C with primary antibody. The antibodies to p-PI3K, p-ERK, p-JNK, p-p38, p-NF-κB p65, p-Src, p-Pyk2, integrin β3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody to F-actin was obtained from Abcam (Cambridge, MA, USA). An antibody to paxillin was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). All primary antibodies were used at 1:1000 dilution. After washing, the nitrocellulose membranes were incubated at RT for 1 h with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and developed using SuperSignal West Pico Chemiluminescent Substrate (Life Technologies Grand Island, NY, USA). Digital images and protein densitometry were analyzed with a G-BOX chemiluminescence imaging system (Syngene, Frederick, MD, USA).