β actin antibody

(R)‐salbutamol (>99% purity, 99.85% ee) and (S)‐salbutamol (>99% purity, 92.73% ee) were provided by Dongguan Key‐Pharma Biomedical Co., Ltd. LPS (Escherichia coli O111:B4), ICI‐ 118551 hydrochloride, fluorescent probes 3‐Amino,4‐aminomethyl‐2′,7′‐difluorescein diacetate (DAF‐FM DA) and 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) were bought from Sigma Chemical Co. The kits for cDNA synthesis, BCA protein assay, cell culture reagents and SYBR Green Supermix were from by Life Technologies Inc (Gibco). Methanol and acetonitrile were acquired from Fisher Chemical. Phycoerythrin (PE)‐conjugated anti‐mouse F4/80 (123110), fluorescein isothiocyanate (FITC)‐conjugated anti‐mouse CD206 (141704) and allophycocyanin (APC)‐conjugated anti‐mouse CD11c (117310) were procured from BioLegend. β‐actin antibody (# BF01980) was obtained from Affinity Biosciences. Inducible nitric oxide synthase (iNOS) mouse antibody (2982S) was obtained from Cell Signaling Technology. The enzyme immunoassay kits for MCP‐1, IL‐1β and TNF‐α were manufactured by Neobioscience. Beyotime Institute of Biotechnology supplied the Cell Counting Kit‐8 (CCK‐8). Rotenone/antimycin A, carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin came from Seahorse Bioscience (Agilent Technologies, Inc).

Specific pathogen-free (SPF) grade 4-week-old (70-80 g) male SD rats were purchased from Hunan Changsha Tianqin Biotechnology Co., Ltd. Common feed, high-fat feed, and streptozotocin (STZ) were all obtained from Beijing Boai Port Biotechnology Co., Ltd. Cx43 antibody (#AF0137), mTOR antibody (#AF6308), AKT antibody (#AF6261), GAPDH antibody (#T0004), β-actin antibody (#AF7018), LC3 antibody (#AF5402), p62 antibody (#AF5384), goat anti-rabbit IgG (#S0001), and goat anti-mouse IgG (#S0002) were all purchased from Affinity Biosciences (Affinity Biosciences Milwaukee, WI 53224, America). Dapagliflozin was provided by AstraZeneca Pharmaceutical Co., Ltd.

Sodium deoxyribonucleic acid salt (derived from salmon sperm) was purchased from Sigma. The mass concentration of DNA in the sodium deoxyribonucleic acid salt of 0.02 mg/mL was determined by NanoDrop to be 1.25 mg DNA/mg sodium deoxyribonucleic acid sodium salt. Sodium carboxymethyl cellulose (0.5%) was used to prepare suspensions containing DNA with final concentrations of 10 mg/kg (low dose), 50 mg/kg (medium dose), and 300 mg/kg (high dose), respectively. Olive oil was purchased from Hangzhou Guanlai Trading Co. Ltd.; CCl₄ was purchased from Sinopharm Chemical Reagent Co. Ltd, diluted with olive oil. Aspartate aminotransferase (AST) kit, alanine aminotransferase (ALT) kit, superoxide dismutase (SOD) kit, malondialdehyde (MDA) kit, glutathione peroxidase (GSH‐Px) kit, and glutathione (GSH) kit were all purchased from Nanjing Jiancheng Biological Engineering Co. Ltd, while ELISA kits TNF‐α and IL‐6 were purchased from Thermo Fisher Scientific, USA. TUNEL (terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick end labeling) apoptosis kit and bicinchoninic acid (BCA) protein concentration determination kit were purchased from Beijing Solarbio Science & Technology Co. Ltd. β‐Actin antibody was purchased from Affinity and other antibodies were purchased from ProteinTech Group.

The proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride. The membranes were blocked for 1 h at room temperature with 5% milk and incubated with the indicated primary antibodies at 4 °C overnight. After washing with PBST three times for 30 min, the membranes were incubated with secondary antibodies for 1 h. The membranes were washed with PBST three times and visualized by chemiluminescence. We used the following antibodies for the experiments: p-RELA/NFκB p65 antibody (Cat: sc-136548, Santa Cruz Biotechnology, TX, USA), MTA1 (Cat: sc-373765, Santa Cruz), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (Cat: SA00001-2, Proteintech, Wuhan, China), HRP-conjugated Affinipure Goat Anti-Mouse IgG (Cat: SA00001-1, Proteintech), and β-actin Antibody (Cat: AF7018, Affinity, Jiangsu, China). Band intensities were quantified by ImageJ and were normalized using β-actin as a housekeeping protein.

Total protein of cells was extracted by RIPA lysis buffer, separated by SDS-PAGE and subsequently transferred to PVDF membranes (Millipore). Membranes were blocked with 5% skim milk at room temperature for 1 h and subsequently incubated with the primary antibody anti-IGF1R (1:1000, Abcam, United States) and β-actin antibody (1:1000, Affinity, United States) at 4°C overnight. A secondary antibody (CST) was used and detected by chemiluminescence.