Figure 7. Assembly of the hTRPV1^N331K channel.

(A) T-REx-293 cell lines stably expressing hTRPV1^WT and hTRPV1^N331K were transfected with hTRPV1^WT-GFP and hTRPV1^N331K-GFP, respectively, and immunoprecipitated using an anti-GFP antibody. Western blot analysis using anti-TRPV1 antibody was performed on the immunoprecipitates (left). Western blot analysis using an anti-TRPV1 antibody (middle) or an anti-GFP antibody (right) was performed on the total lysate (input). An anti-actin antibody was used as a positive control (n = 3). Note that both hTRPV1^WT and hTRPV1^N331K were pulled down by the anti-GFP antibody only when expressed together with hTRPV1^WT-GFP and TRPV1^N331K-GFP, respectively. (B) Western blot analysis of total cell lysates from T-REx-293 cells stably expressing hTRPV1^WT or hTRPV1^N331K in seminative conditions, using an anti-TRPV1 antibody at different temperatures and incubation durations (n = 4, see Methods). Note that the intensity of the higher-molecular-weight bands was reduced or disappeared when lysates were incubated at 95°C or when DTT was added, respectively, while the low-molecular-weight band became stronger. (C) BS³ crosslinker promoted tetramerization in both hTRPV1^WT and hTRPV1^N331K channels. An anti-TRPV1 antibody was used in Western blot analysis of total cell lysates from T-REx-293 cells stably expressing hTRPV1^WT or hTRPV1^N331K and incubated with different doses of the crosslinker BS³ in seminative conditions (n = 3, see Methods). Note that the higher-molecular-weight band intensities increased as the BS³ dose increased.