Fig. 3: The multimerization, but not intracellular N-C binding of TRPM8, regulated by LCK.

A–D The effect of LCK on the intracellular N-C binding of TRPM8. A, B Expression constructs for Flag-tagged N-terminus of TRPM8 (Flag-M8N) and GFP-M8C were co-transfected with HA-LCK or control vector into HEK293T cells, before harvest for treatment with 10 μM PP2 for 24 h. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the intracellular N-C binding of TRPM8. C, D Similar experiments in (A) and (B) but cells co-expressing Flag-M8N and GFP-M8C along with siLCK. E–L The effect of LCK on the multimerization of TRPM8. E, F Expression constructs for Flag-TRPM8 were co-transfected with HA-LCK or control vector into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min, a crosslinking agent. The cell lysates were subjected to WB assay with the indicated antibodies to detect the level of TRPM8 multimerization. G, H Similar experiments in (E) and (F) but cells co-expressing Flag-TRPM8 along with siLCK. I, J Flag-TRPM8 and GFP-TRPM8 were co-transfected with or without HA-LCK into AsPC-1 cells. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of intermolecular TRPM8. K, L Similar experiments in (I) and (J) but cells co-expressing Flag-TRPM8 and GFP-TRPM8 along with siLCK. **P < 0.01, ***P < 0.001, NS not significant. Data are presented as mean ± SEM. All studies were repeated at least three times.