Ecl a and b

MEECs were treated with 17β-estradiol at the concentrations of 1 nM, 10 nM, 100 nM for 24 h. Cell lysates were collected for western blotting assay. The protein samples were specifically probed with primary antibodies: mouse anti-ERα (1:1000, Santa Cruz Biotechnology, sc-71064), mouse anti-PR (1:1000, Santa Cruz Biotechnology, sc-398898), and mouse anti-β-actin (1:1000, Santa Cruz Biotechnology, sc-47778) 4 °C overnight and then conjugated with the following secondary antibodies: m-IgGκ BP-HRP (1:1000, Santa Cruz Biotechnology, sc516102). Immunoblots were colorated with a mixture of enhanced chemiluminescence reagents ECL A and B (Beyotime Biotechnology) at a ratio of 1:1 and digitally photographed in UV trans-illuminator (Bio-Rad Laboratories).

HNVEC cells were added with or without a low dose of 0.5 nM actinomycin D (Act D) for 24 hrs and then scratched using a cell scraper into 200μl modified radioimmuno-precipitation assay (RIPA) buffer containing protease inhibitors as described in early study [15 (link)] with HeLa cells as a control. The collected protein samples were measured concentration by BCA (Beyotime Biotechnology, Shanghai, China) method using microplate spectrophotometer (Bio-Tek, Vermont, USA). SDS polyacrylamide gels electrophoresis was conducted and then transferred electrophoretically on to a 0.2μm polyvinylidenedifluoride (PVDF) membranes (Immobilon-NC, Millipore, Italy), specifically probed with following primary antibodies: mouse anti-p53 (1:1000, Santa Cruz Biotechnology, CA, USA, sc-126), rabbit anti-p21 (1:1000, Santa Cruz Biotechnology, CA, USA, sc-397) and mouse anti-β-actin (1:100, Proteintech Group, Inc., Chicago, USA) 4°C overnight and then conjugated with the following secondary antibodies: HRP-labeled goat anti-mouse/rabbit lgG (Santa Cruz Biotechnology, CA, USA). Immunoblots were colorated with an mixture of enhanced chemiluminescence reagents ECL A and B (Beyotime Biotechnology, Shanghai, China) at a ratio of 1:1 and digitally photographed in UV trans-illuminator (Bio-rad Laboratories, California, USA).