Fig. 4
ERK2 phosphorylates transgelin-2 at S145 residue. a. The sequence of transgelin-2 in different species. b. In vitro kinase assay was performed using purified Flag-ERK2 and GST-transgelin-2. Phosphorylation of transgelin-2 at S145 was detected by anti-phospho-serine (p-ser) and anti-phospho-S145-transgelin-2 (p-T2-S145) antibodies. c. SW1990 cells were transfected with wild-type (W), 29-31aa truncated mutant (∆) or S145A mutant (A) of GFP-tagged transgelin-2 for 48 h. Phosphorylation of transgelin-2 on S145 was detected by anti-phospho-S145-transgelin-2 (p-T2-S145) antibody. d. KRAS wild-type cells (Hs 766 T, BxPc3 and Hs 700 T) and mutant cells (SW1990 Capan-1, and PANC-1) were collected for western blot assay. Phosphorylation of transgelin-2 on S145 was detected by anti-phospho-S145-transgelin-2 (p-T2-S145) antibody. e. HPDE6-C7 cells were transfected with KRASG12D mutant or vector control for 48 h. The cells were then treated with 15uM U0126 for 6 h. Phosphorylation of transgelin-2 on S145 was detected by anti-phospho-S145-transgelin-2 (p-T2-S145) antibody. f. SW1990 cells were treated with ERK1/2 inhibitor U0126 for 6 h or PI3K inhibitor LY294002 for 3 h. Phosphorylation of transgelin-2 on S145 was detected by anti-phospho-S145-transgelin-2 (p-T2-S145) antibody