Western blotting detection system

Methoxy-stilbenes, 3,4,2′-trimethoxy-trans-stilbene (3MS), 3,4,2′,4′-tetramethoxy-trans-stilbene (4MS), and 3,4,2′,4′,6′-pentamethoxy-trans-stilbene (5MS), were synthesized in the Department of Chemical Technology of Drugs, PUMS, as described previously [5 (link)]. Resveratrol, dithiothreitol, antibiotics solution (10⁴ U penicillin, 10 mg streptomycin, 25 μg amphotericin B), bovine serum albumin, dimethylsulfoxide (DMSO), Dulbecco’s Modified Eagle’s Medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RIPA buffer, trypsin, Tris, and tRNA from E. coli were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Primary antibodies against ERα, CYP19, CYP1A1, and CYP1B1 were obtained from Abgent (San Diego, CA, USA). Primary antibodies against AhR, β-actin, and secondary antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protease inhibitor tablets were obtained from Roche Diagnostics GmbH (Penzberg, Germany). SDS-PAGE Gels (7.5, 10, and 12%) and the Western blotting detection system were purchased from Bio-Rad Laboratories (Hercules, CA, USA). All other compounds were readily available commercial products. Resveratrol and its methoxy derivatives were dissolved in DMSO at the concentration of 100 mM and stored at − 20 °C.

For both mouse and human samples, equal amounts of total protein from LV myocardial tissues were electrophoresed on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies overnight at 4 °C, washed, and then incubated with secondary antibodies for one hour. Immunoreactive bands were detected with SuperSignal West Femto (Thermo Fisher Scientific, Waltham, MA, USA) in a Western blotting detection system (Bio-Rad Laboratories, Hercules, CA, USA). Results are expressed as density values normalized to GAPDH.

The liver tissues were homogenized in radioimmunoprecipitation assay lysis buffer (1% Triton X-100, 1% deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)), and protein concentration was measured. Equal amounts of protein extracts were mixed (3 : 1, v/v) and processed in loading buffer for electrophoresis in 10% acrylamide SDS gels and subsequently electroblotted to a nitrocellulose transfer membrane (Merck Millipore, Burlington, MA, USA) using a Trans-Blot SD semidry electrophoretic transfer cell (Bio-Rad). Target proteins were probed with specific primary antibodies, and then, the bound primary antibodies were recognized with species-specific secondary antibodies. The chemiluminescence intensity of the specific proteins on the membrane was subsequently detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and a western blotting detection system (Bio-Rad). The optical densities (OD) of the bands were quantified using the Gel-Pro 3.0 software (Biometra, Goettingen, Germany). The density of the specific protein band was corrected to eliminate background noise and normalized to that of GAPDH (Boster Biological Technology Ltd., Wuhan, China) as OD/mm².

Total protein was obtained from the ventricular myocardial tissues using tissue homogenates, centrifugation and heat denaturation. The protein lysates were electrophoresed and separated by 6–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore Immobion-P; BioSharp, Anhui, China). The membranes were blocked with 5% skim milk at room temperature for 1 h, and then incubated overnight at 4°C with primary antibodies, including rabbit anti-Bcl-2 (BA0412; 1:500), rabbit anti-Bax (BA0315-2; 1:500), rabbit anti-Calr (BM1798; 1:500), rabbit anti-GRP78 (BA2042; 1:500) (all from Boster Biotechnology, Inc., Wuhan, China), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (AP0063; 1:5,000; BioWorld, Inc., Jiangsu, China). The membranes were then incubated with IRDye800-conjugated secondary antibodies (1:20,000; Rockland, Inc., Gilbertsville, PE, USA) at room temperature for 1 h. The Odyssey double color infrared laser imaging system (LI-COR; Lincoln, NE, USA) was used to detect the antigen-antibody complexes in a western blotting detection system (Bio-Rad). The results were expressed as density values normalized to GAPDH.

Proteins were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane. The membranes were blocked with Ncm Blot Blocking Buffer for 15min at room temperature, followed by incubation with anti-SMAD2 (67343-1-Ig, proteintech), anti-Phospho-SMAD2 (#3108, Cell Signal Technology), anti-GSDMD (20770-1-AP proteintech), anti-ASC (67494-1-Ig, proteintech), anti-NLRP3 (#13158, Cell Signaling Technology) and GAPDH antibody (60004-1-Ig, proteintech) overnight at 4 °C. The membranes were washed three times and incubated with secondary anti-rabbit IgG (SA00001-2, proteintech) or anti-mouse IgG (SA00001-1, proteintech) for 1 h. Protein bands were visualized on a Western blotting detection system (Bio-Rad, USA).

After different doses (described above) of drug treatment for 48 h, cells (HT29, SW837, LS180, SW480 and HCT116) were lysed in RIPA lysis and extraction buffer (Cat# 89,900, Thermo, USA). BCA protein assay kits (Beyotime, China) were used for collection and quantification of proteins. Electrophoresed by 10% SDS‒PAGE, proteins were moved to a PVDF membrane (Bio-Rad, USA) with transfer buffer. The 1-hour blocking of membranes was made with 5% nonfat milk powder, followed by incubation with primary antibodies (1:1000) against β-actin (Cell Signaling Technology (CST), #3700), PI3K (Proteintech, 60225-1-Ig), P-PI3K (CST, #17,366), ERK1/2 (Proteintech, 67170-1-Ig); P-P44/42MAPK (ERK1/2) (CST, #4370), AKT (CST, #4691), P-AKT (CST, #4060), MEK1/2 (Proteintech, 110499-1-AP), and P-MEK1/2 (CST, 9154) overnight at 4 °C. The next day, the 1-hour incubation of membranes was made with the appropriate secondary antibodies (goat anti-rabbit IgG/HRP (1:10000, ZSGB-BIO, ZB-2301) or goat anti-mouse IgG/HRP (1:10000, ZSGB-BIO, ZB-5305)) at room temperature. A western blotting detection system (Bio-Rad, USA) was adopted to visualize protein bands.

Cultured H9c2 cells were harvested by scraping and centrifugation, washed with PBS, and re-suspended in RIPA buffer. Soluble proteins were collected by centrifugation at 12,000 g. Protein lysates were subjected to 10% and 12% SDS-PAGE and transferred onto a NC membrane (Millipore Corporation). After blocking with 5% skim milk, the membranes were incubated with the respective primary antibodies (caspase-3, 1:1000; caspase-9, 1:1000, Apaf-1, 1:1000 Cell Signaling Technology, USA) in Tris-buffered saline (TBS) containing 0.1% Tween-20 overnight at 4 °C. The membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated IgG antibodies at a 1:5000 dilution (Proteintech Group Inc., USA). Immunoreactive proteins were then visualized using ECL. The signals were quantified by densitometry using a Western blotting detection system (Bio-Rad Laboratories, Inc, USA). Actin served as the loading control.