Fig. 5: 14-3-3ζ interaction with TRPM8 for regulating TRPM8 multimerization.

A GST pull-down assay. Purified GST alone or GST-M8C fusion proteins expressed in E. coli BL21 bacteria were incubated with the lysates from HEK293T cells expressing GFP-14-3-3ζ constructs and subjected to WB assay. B Co-IP assay. The lysates of native PANC-1 cells were added to an anti-14-3-3 antibody for IP and then subjected to WB assay with an anti-TRPM8 antibody. C, D Expression constructs for Flag-TRPM8 with or without GFP-14-3-3ζ were transfected into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min. The lysates were subjected to WB assay with the indicated antibodies to detect the level of TRPM8 multimerization. E, F Similar experiments in (C) and (D) but cells co-expressing Flag-TRPM8 along with human 14-3-3ζ-specific siRNAs (si14-3-3ζ#1, #2, or #3). G, H AsPC-1 cells were co-transfected Flag-TRPM8 and GFP-TRPM8 with or without si14-3-3ζ, and then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of intermolecular TRPM8. **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM. All studies were repeated at least three times.