Figure 5. Tyrosine phosphorylation of ITSN2 depends on mitogenic stimulation and clathrin-mediated endocytosis.

HeLa cells were maintained with 10% FBS, starved for 16 h or starved for 16 h and treated with 20 ng/ml of EGF for 10 and 20 min (A, B). For inhibition of endocytosis, serum-starved cells were treated with 30 µM chlorpromazine (CPZ) for 20 min followed by stimulation with EGF (20 ng/ml) in the presence of chlorpromazine (C). Extracts of cells were subjected to immunoprecipitation with antibodies against ITSN2 (A, C) and ITSN1 (B). Normal rabbit serum (NRS) was used as control. The precipitated proteins were analyzed by Western blotting with anti-phosphotyrosine, anti-ITSN2 and anti-ITSN1 antibodies. The ratio of pITSN2/ITSN2 intensity values was calculated, the data are presented as mean ±SEM (n = 3);*P<0.05,**P<0.01,***P<0.001. IP, immunoprecipitation; WB, Western blotting.