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. 2020 Dec 4;10:573127. doi: 10.3389/fonc.2020.573127

Figure 6.

Figure 6

AMPK interacts with TRPM8. (A, B) Co-IP analysis. (A) Constructs for GFP-TRPM8 and Flag-AMPK expression were transiently transfected into MCF7 cells. After 48 h of transfection, protein lysates were immunoprecipitated with an anti-GFP antibody and assayed by immunoblot with an anti-Flag antibody (lower panel). Reciprocal Co-IP with an anti-Flag antibody used for immunoprecipitation and anti-GFP used for WB analysis (upper panel) (N = 3). (B) The construct for GFP-AMPK expression was cotransfected with M8-N, M8-LI, M8-LII, or M8-C into MCF7 cells. Protein lysates were immunoprecipitated with an anti-Flag antibody and assayed by immunoblot with an anti-GFP antibody (upper). The constructs for GFP-AMPK and Flag-M8-C expression were cotransfected into MCF7 cells. Protein lysates were immunoprecipitated with an anti-GFP antibody and assayed by immunoblot with an anti-Flag antibody (upper) (N = 3). (C) GST pull-down analysis. Protein lysates of MCF7 cells transiently expressing Flag-AMPK were incubated with purified cytoplasmic C-terminus of TRPM8 GST fusion protein (GST-M8C). GST-M8C, but not control GST, successfully pulled down Flag-AMPK. PD: pull-down. The lysate was used as a positive control (N = 3). N represents the number of replicate experiments. PD, pull down; WCL, whole cell lysates.