Anti notch1

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).

For the co‐immunoprecipitation assay, ice‐cold PBS was first used to wash the cells, following which they were transfected with the NEDD4‐carrying vector. After transfection, the cells were lysed in lysis buffer supplemented with phosphatase and protease inhibitors. Next, the cell lysates were incubated first with the primary antibodies and then with immunoprecipitation IgG beads. The immunoprecipitates were washed with lysis buffer and boiled for 5 min in protein loading buffer. Finally, the co‐immunoprecipitated proteins were identified by Western blot assay using anti‐NEDD4 (Proteintech) and anti‐Notch1 (Proteintech) primary antibodies. For the ubiquitination assay, HA‐ubiquitin and S‐Tag‐Notch1 were added to cells that had been transfected with either the NEDD4‐carrying vector or the empty vector. The cells were then treated with 10 μM MG‐132 for 48 h before harvesting. The harvested cells were then subjected to immunoprecipitation with S‐Tag‐beads and immunoblotting as described above, with anti‐ubiquitin (Santa Cruz Biotechnology) and anti‐β‐actin (Proteintech) used as the primary antibodies for the Western blot assay.