Spectra multicolour high range protein ladder

Protein was extracted from HUVECs by RIPA solution (P0013B, Beyotime) combined with phenylmethanesulfonyl fluoride (PMSF) and a phosphatase inhibitor. Then, all the protein samples were mixed with 1X SDS–PAGE protein loading buffer (P0015A, Beyotime). After all the samples were denatured at 100°C for 10 min, electrophoresis was performed using a 10% SDS–PAGE gel preparation (Bio-Rad, USA) at 80 V for 30 min and 120 V for 90 min. Then, the proteins were blotted onto a di-fluoride polyvinylidene fluoride (PVDF) membrane at 240 mA for 2 h in ice water. Next, the PVDF membrane was blocked in QuickBlock™ buffer (P0252, Beyotime) for 40 min and then incubated with diluted primary antibodies (Table 3) at 4°C overnight. After washing three times with tris buffered saline with tween (TBST), horseradish-peroxidase-conjugated (HRP-conjugated) antibodies were added to the PVDF membrane. After three washes, the immunoreactive bands were visualized using ChemiDoc Touch (Bio-Rad, USA). We used a Spectra multicolour high-range protein ladder (26 625, Thermo Scientific) as the protein ladder.

All samples were boiled in 1 × Laemmli buffer and subsequently separated by gel electrophoresis in NuPAGE 3–8% Tris–acetate SDS polyacrylamide gradient gels (Invitrogen) together with Spectra Multicolour High Range Protein Ladder (Thermo Fisher) and HiMark Prestained Protein Standard (life technologies) as range indicators. Separated proteins were transferred to nitrocellulose membranes. After blocking with 5% block milk, membranes were incubated with corresponding primary and HRP-bound secondary antibodies in 5% block milk. Visualization was performed using ECL reaction (Western Blotting Luminol, Santa Cruz Biotechnology) on X-Ray Film (Fuji Medical Super RX).