Ecl chromogenic agent

HC-A cells or the cartilage tissue of rats were harvested, rinsed with cold PBS three times, and added to 100~200 μL RIPA lysate (Beyotime Biotechnology, Shanghai, China). The cells underwent ultrasonic water-splitting, and the protein content was quantified by the Bradford method. The same amount of protein in each group was subjected to 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). Afterward, the primary antibodies (1:1000) of Bax (ab32503), cleaved-Caspase3 (ab2302), NLRP3 (ab214185), ASC (ab180799), cleaved Caspase-1 (ab74279), MMP3 (ab52915), MMP13 (ab39012), SIRT1 (ab189494), FoxO3a (ab109629), NF-ĸB p65 (ab207297), p-NF-ĸB p65 (ab239882), and GAPDH (ab181602) were added and incubated at 4° C overnight. After the membranes were cleaned twice with TBST, they were incubated at room temperature for 1 hour with fluorescein-labeled Goat anti Rabbit IgG (ab205718,1:2500). The above antibodies were obtained from Abcam (Cambridge, UK). At last, the membranes were rinsed three times, exposed with enhanced chemiluminescence (ECL) chromogenic agent (Millipore, Bedford, MA, USA), and captured with a membrane scanner.

The cells were treated, and the culture medium was removed. Then, the protein lysate (Roche) was added for total protein separation. 50 μg total protein was subjected to 12% polyacrylamide gel and went through 2 h of electrophoresis at 100 V. It was then transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skimmed milk at RT for 1 h, the membranes were rinsed with TBST three times (10 min each) and incubated with the primary antibodies (1:1,000) of anti-Bax (ab182734), anti-P53 (ab32389), anti-Caspase3 (ab32351), anti-P21 (ab109199), anti-HIF-1α (ab179483), anti-HECTD2 (ab249770), anti-E-cadherin (ab40772), anti-Vimentin (ab92547), and anti-N-cadherin (ab76011) overnight at 4°C. After washing the membranes with TBST, we incubated them with horseradish peroxidase (HRP)-labeled Goat anti-Rabbit IgG (ab205718) (1:2,500) for 1 h at RT. The above antibodies were obtained from Abcam (Cambridge, United Kingdom). Afterward, the membranes were cleaned with TBST 3 times (10 min each). Finally, the ECL chromogenic agent (Millipore, Bedford, MA, United States) was employed for exposure and imaging with a membrane scanner.

After cell treatment, the medium was discarded. Supplementation of protein lysis solution (Roche) was completed for separating the total protein. Then, 50 μg of total protein was sampled on 12% polyacrylamide gel for 2-hour electrophoresis at 100 V. Afterward, the electrophoresed total protein was transferred to polyvinylidene fluoride (PVDF) membranes. One hour after being sealed with 5% skimmed milk at RT, the membranes were flushed three times (10 minutes each time) with Tris-buffered saline with Tween-20 (TBST) for overnight incubation at 4°C with the following primary antibodies (Abcam, Cambridge, UK, concentration 1:1000): Anti-Bax antibody (ab182734), Anti-Bcl-2 antibody (ab692), Anti-Cleaved Caspase-3 antibody (ab214430), Anti-p-AKT antibody (ab38449), Anti-AKT antibody (ab8805), Anti-p-PI3K antibody (ab278545), Anti-PI3K antibody (ab140307), and Anti-HIF-1 alpha Antibody (ab179483). Next, the membranes were maintained with the horseradish peroxidase (HRP)-labeled Goat Anti-Rabbit IgG (ab6205718) (concentration 1:2500) at RT for one hour after being rinsed with TBST. Following 3 washes with TBST (10 minutes/time), the membranes were exposed with enhanced chemiluminescence (ECL) chromogenic agent (Millipore, Bedford, MA, USA), and a membrane scanner was utilized for imaging [24 (link)].