Figure 5.
Figure 5. Rap1 is a TRPM8-interacting protein. (A) Representative immunoprecipitation experiments. Expression vectors encoding Rap1-WT-HA were transfected into HEK-overexpressing TRPM8- or TRPM8Y905A-tagged cells. Cell lysates were immunoprecipitated (IP) with an anti–HA antibody and immunoblotted with antibodies against TRPM8 and HA. Images in C and D are representative of three independent experiments. (B) In situ detection of endogenous TRPM8/Rap1 interaction in HMECs. TRPM8/Rap1 complexes were monitored using PLA using anti–TRPM8 and Rap1 antibodies followed by staining with proximity probes, ligation, and localized rolling-circle amplification. HMECs silenced for TRPM8 (siTRPM8) or not (siCNTRL), in the presence or absence of 10 µM icilin (10 min treatment). Close locations between the two proteins were observed as red fluorescent dots and DAPI-stained nuclei as blue. (Bii) Puncta density quantified as mean ± SEM puncta per cell. *, P ≤ 0.05; **, P ≤ 0.001 (Student’s t test). (Ci) TRPM8 N-terminal tail (GST-Nt), C-terminal tail (GST-Ct), or GST were incubated with lysates of HEK cells overexpressing Rap1-WT-GFP, Rap1-N17-GFP, or Rap1-V12 and precipitated using GST. Western blotting was performed with anti–GFP antibody. One representative experiment of three is shown. (Cii) Quantification of Rap1/TRPM8 tails normalized over the input. Data are expressed as mean ± SEM. *, P < 0.05 (Student’s t test). Inset shows a scheme representing GST-Nt and C-terminal tail purification. (D) GST pull-down assay of in vitro translated Rap1WT, Rap1-N17, or Rap1-V12 using FluoroTect and GST or GST fused to the GST-Nt or GST-Ct. 10% of the in vitro translated Rap1 were used for the input of the GST pull-down (top). Bodipy-FL-stained gel are shown for the expression of Rap1. (E) The GST pull-down assay was repeated by loading in vitro translated RAP1-WT with 1 mM GDP (left) or 0.1 mM GTP (right). Anti-GST or anti–Rap1 antibodies were used to visualize the interaction of Rap with GST-fused N- and C-terminal tail. All the gels are representative of at least three independent experiments.

Rap1 is a TRPM8-interacting protein. (A) Representative immunoprecipitation experiments. Expression vectors encoding Rap1-WT-HA were transfected into HEK-overexpressing TRPM8- or TRPM8Y905A-tagged cells. Cell lysates were immunoprecipitated (IP) with an anti–HA antibody and immunoblotted with antibodies against TRPM8 and HA. Images in C and D are representative of three independent experiments. (B) In situ detection of endogenous TRPM8/Rap1 interaction in HMECs. TRPM8/Rap1 complexes were monitored using PLA using anti–TRPM8 and Rap1 antibodies followed by staining with proximity probes, ligation, and localized rolling-circle amplification. HMECs silenced for TRPM8 (siTRPM8) or not (siCNTRL), in the presence or absence of 10 µM icilin (10 min treatment). Close locations between the two proteins were observed as red fluorescent dots and DAPI-stained nuclei as blue. (Bii) Puncta density quantified as mean ± SEM puncta per cell. *, P ≤ 0.05; **, P ≤ 0.001 (Student’s t test). (Ci) TRPM8 N-terminal tail (GST-Nt), C-terminal tail (GST-Ct), or GST were incubated with lysates of HEK cells overexpressing Rap1-WT-GFP, Rap1-N17-GFP, or Rap1-V12 and precipitated using GST. Western blotting was performed with anti–GFP antibody. One representative experiment of three is shown. (Cii) Quantification of Rap1/TRPM8 tails normalized over the input. Data are expressed as mean ± SEM. *, P < 0.05 (Student’s t test). Inset shows a scheme representing GST-Nt and C-terminal tail purification. (D) GST pull-down assay of in vitro translated Rap1WT, Rap1-N17, or Rap1-V12 using FluoroTect and GST or GST fused to the GST-Nt or GST-Ct. 10% of the in vitro translated Rap1 were used for the input of the GST pull-down (top). Bodipy-FL-stained gel are shown for the expression of Rap1. (E) The GST pull-down assay was repeated by loading in vitro translated RAP1-WT with 1 mM GDP (left) or 0.1 mM GTP (right). Anti-GST or anti–Rap1 antibodies were used to visualize the interaction of Rap with GST-fused N- and C-terminal tail. All the gels are representative of at least three independent experiments.

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