Pab36269

Total protein content was extracted from 3 × 105 cells in each group using radioimmunoprecipitation assay lysis buffer (Bioswamp) and quantified using a bicinchoninic acid kit (Bioswamp) following the manufacturer's protocol. 20 μg of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies against CDC2 (Bioswamp, PAB30052, 1 : 1,000), B-cell lymphoma-2 (Bcl-2, Bioswamp, PAB30042, 1 : 1,000), Bcl-2-associated X (Bax, Bioswamp, PAB30040, 1 : 1,000), nuclear factor erythroid-2-related factor 2 (Nrf2, Bioswamp, PAB30175, 1 : 1,000), TrxR1 (abcam, ab124954, 1 : 5,000), apoptosis signal regulating kinase 1 (ASK1, Bioswamp, PAB36297-P, 1 : 1,000), phosphorylated (p)-ASK1 (abcam, ab47304, 1 : 1,000), and GAPDH (Bioswamp, PAB36269, 1 : 1,000) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-labeled goat antirabbit IgG secondary antibody (Bioswamp, PAB160011, 1 : 20,000) for 1 h at room temperature and visualized using a Tanon-5200 apparatus (Tanon, Shanghai, China). The band gray values were read using the TANON GIS software (Tanon). GAPDH acted as the internal reference.

Total proteins were extracted from VSMCs using a radioimmunoprecipitation assay lysis buffer (R0010; Solarbio, China) and quantified using a bicinchoninic acid kit (PC0020; Solarbio, China). Aliquots (20 µg) of the extracted proteins were separated on polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (IPVH00010; Millipore, USA). Having initially blocked the membranes, these were then incubated for 1 h with primary antibodies against RUNX2 (PAB33483), Smad7 (PAB40077), Wnt3a (PAB30170), β-catenin (PAB30715), and GAPDH (PAB36269) at a dilution ratio of 1:1000, all of which were purchased from Bioswamp (Wuhan, China). Thereafter, the membranes were incubated with a goat anti-rabbit IgG secondary antibody (SAB43714; Bioswamp, China) for 1 h.

Protein was extracted and quantified using a BCA protein assay kit (Beyotime, China). Total protein was separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with a buffer containing 5% nonfat milk in PBS with 0.05% Tween-20 for 2 h and incubated with the primary antibody: anti-STAT3 antibody (1 : 1000, PAB30641, Bioswamp), anti-p-STAT3 (1 : 2000, ab76315, Abcam), anti-JAK (1 : 2000, PAB30711, Bioswamp), anti-p-JAK (1 : 2000, ab108596, Abcam), and anti-GAPDH (1 : 1000, PAB36269, Bioswamp) antibody. After three washes with PBS/Tween-20, the membranes were incubated with horseradish peroxidase-conjugated secondary goat antirabbit IgG (1 : 20000, SAB43714, Bioswamp) for 2 h at 25°C. Protein bands were visualized by enhanced chemiluminescence color detection (Tanon-5200, TANON, China) and analyzed using the TANON GIS software.

Total proteins were extracted from harvested cells using radioimmunoprecipitation assay lysis buffer (Bioswamp) and quantified using a bicinchoninic acid kit (Bioswamp) according to the manufacturer’s instructions. Proteins (20 μg) were separated and transferred on to polyvinylidene fluoride membranes (Millipore, MA, U.S.A.), followed by blocking with skim milk. The membranes were then incubated with primary antibodies against AMPKα1 (Bioswamp, PAB30970, 1:1000), p-AMPKα1 (Bioswamp, PAB36316-P, 1:1000), glucose transporter 1 (GLUT1, Bioswamp, MAB37348, 1:1000), hexokinase 1 (HK1, Bioswamp, MAB37234, 1:1000), lactate dehydrogenase A (LDHA, Bioswamp, PAB30703, 1:1000), and GAPDH (Bioswamp, PAB36269, 1:1000) overnight at 4°C, followed by incubation with goat anti-rabbit IgG secondary antibodies (Bioswamp, SAB43714, 1:20000) for 1 h at room temperature. GAPDH served as the internal reference.

The herbal WYHZTH formula was decocted by the Traditional Chinese Medicine Laboratory Center of the First Affiliated Hospital of Guangxi University after the identification of crude drugs was confirmed. Thymopentin injection (M107827, Aladdin), 20% human serum albumin (HSA), D-galactosamine (D-Gal), and lipopolysaccharide (LPS) were purchased from Sigma. The other reagents used were as follows: hematoxylin-eosin (Cat. No. E8090, G1140, G8590, Solarbio); protein marker, BCA protein concentration determination kit (Cat. No. XY-MY-0112, XY-MY-0096, Shanghai Xuanya), PVDF membrane, ECL luminescence reagent (Cat. No. XF- P3360, ZDSJ140, Xinfan Company), Tween-20 (Cat. No. PW0028, LEAGEN Company); RIPA tissue cell rapid lysate (Cat. No. BL504A, Biosharp); Nephrin, Podocin, LC3-I/II, Beclin1, P62, AMPK, and GAPDH protein primary antibody (Cat. Nos. PAB40854, PAB44275, PAB34124, PAB44768, PAB35470, PAB30970, and PAB36269, Bioswamp); p-AMPK and p-ULK1 (Cat. Nos. 50081S and 14202S, CST); goat anti-rabbit IgG (Cat. No. SAB43714, Bioswamp); and MaxVision TM secondary antibody and HRP-polymer (Cat. No. Kit-5020, Maixin).