Anti notch3

Cells were lysed using RIPA buffer, and BCA protein assay kit (Beyotime, Shanghai, China) was utilized to calculate the protein concentration. Then cellular proteins were subjected to SDS‐PAGE gels and transferred to polyvinylidene difluoride membrane (Millipore, USA). Blots were blocked with 5% nonfat milk for an hour, before incubating with primary antibodies overnight at 4°C followed with appropriate secondary antibodies for an additional an hour at room temperature. The protein band was detected by ChemiDoc™ XRS + system and Image J software were applied for determination of the protein abundance. Primary antibodies used in Western blot analyses included anti‐GHITM (Proteintech, China, 16,296‐1‐AP), anti‐YY1 (Proteintech, China, 22156‐1‐AP), anti‐PD‐L1 (Proteintech, China, 28076‐1‐AP), anti‐Notch1 (Proteintech, China, 20687‐1‐AP), anti‐Notch2 (Proteintech, China, 28580‐1‐AP), anti‐Notch3 (Proteintech, China, 55114‐1‐AP), anti‐Notch4 (Abcam, UK, ab184742) and anti‐GADPH (Proteintech, China, 10494‐1‐AP).

Western blot analysis was performed as previously described25 (link). The primary antibodies were as follows: mouse monoclonal anti-E-cadherin (1:2000, Proteintech, Rosemont, USA), mouse monoclonal anti-Vimentin (1:10000, Proteintech), rabbit polyclonal anti-MMP9 (1:500, Proteintech), rabbit polyclonal anti-VEGF (1:1000, Proteintech), rabbit polyclonal anti-ELF3 (1:500, Abcam, Cambridge, MA), rabbit polyclonal anti-Notch3 (1:500, Proteintech). Rabbit polyclonal anti-GAPDH (1:5000, Proteintech) was used as an endogenous control.