Chemidoc touch image analyzer

Total protein was extracted and size-fractioned by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% skim milk in PBS with 0.1% Triton-X100, immunodetection was carried out using specific primary antibodies: anti-VCAM (Abcam), anti-ICAM (BioLegend), anti-p50 (Santa Cruz Biotechnology), anti-p84 (Santa Cruz Biotechnology), anti-p65 (Santa Cruz Biotechnology, Dallas, TX), anti-FSP-1 (Abcam), anti-CD31 (Abcam), anti-VE-Cadherin (Abcam), anti-α-SMA (Sigma-Aldrich).Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology); was used as loading control. Thereafter, blots were incubated with HRP-labeled respective (anti-mouse or anti-rabbit) secondary antibodies (Santa Cruz Biotechnology), washed and processed with Clarity™ Western ECL Substrate (Thermo Fisher Scientific). Signal was detected using ChemiDoc™ Touch image analyzer (Bio-Rad Laboratories).

Total protein from EndMT-undergoing HUVECs was extracted and size-fractioned by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% skim milk in PBS with 0.1% Triton-X100, immunodetection was carried out using specific primary antibodies: anti-p-p65 (Cell Signaling, Danvers, MA), anti-p65 (Santa Cruz Biotechnology), anti-p-Smad2 (Cell Signaling), anti-Smad2 (Cell Signaling), anti-p-p38 (Cell Signaling), anti-p38 (Cell Signaling), anti-p-Erk (Cell Signaling), anti-Erk (Cell Signaling), anti-TWIST (Santa Cruz Biotechnology), anti-FSP-1 (Abcam), anti-CD31 (Abcam), anti-VE-Cadherin (Abcam), anti-α-SMA (Sigma-Aldrich), anti-E-Selectin (Santa Cruz Biotechnology). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology); was used as loading control. Thereafter, blots were incubated with HRP-labeled respective (anti-mouse or anti-rabbit) secondary antibodies (Santa Cruz Biotechnology), washed and processed with Clarity™ Western ECL Substrate (Thermo Fisher Scientific). Signal was detected using ChemiDoc™ Touch image analyzer (Bio-Rad Laboratories).