Fig. 7: LCK activity regulated by TRPM8 phosphotyrosine.

A, B LCK phosphorylation assay. PANC-1 cells were co-transfected HA-LCK with the control vector, WT-TRPM8 or mutant TRPM8-Y1022 with the indicated antibodies as shown in (A), and quantification of the level of LCK phosphorylation shown in (B). C–F LCK ubiquitination assay. C, D Expression constructs for HA-LCK and Myc-Ub were co-transfected with control vector, WT-TRPM8 or mutant TRPM8-Y1022 into AsPC-1 cells. The cells were treated with 10 μM MG132 for 6 h before harvest and used for IP with an anti-HA antibody and WB with the indicated antibodies. E, F Expression constructs for Myc-Ub and WT-TRPM8 or mutant TRPM8-Y1022F were co-transfected with HA-tagged wild-type or mutant LCK into PANC-1 cells. The cells were treated with 10 μM MG132 before harvest and used for IP with an anti-HA antibody and WB with the indicated antibodies. ***P < 0.001, NS not significant. Data are presented as mean ± SEM. All studies were repeated at least three times.