Downregulation of TRPV1 and PKCβII. A, Expression of TRPV1 was downregulated by PKCα and PKCβII, but not by PKCδ or PKCε. HEK 293 cells were transfected with TRPV1–V5 together with different PKC isoforms (all tagged with GFP at their N terminus) as indicated. The expression of TRPV1 was detected with anti-V5 antibody (top blot). The blot was stripped and reprobed for PKCs with anti-GFP, followed by further stripping and reprobing with antitubulin to show similar expression of tubulin and thus similar sample loading in all cases (bottom blot). B, Downregulation of PKCα and βII by TRPV1. HEK 293 cells were transfected with different GFP-tagged PKC isoforms as indicated together with (+) or without (−) TRPV1. The expression of all PKCs was detected by anti-GFP (top blot). The blot was stripped and reprobed with antitubulin to show similar expression in all groups (bottom blot). C, Time course of downregulation of TRPV1 by PKCβII. TRPV1–V5, and PKCβII-pTRE2 were cotransfected in Tet-On HEK 293 cells. Twenty-four hours after transfection, PKCβII was induced by DOX for different times, as indicated. The expression of TRPV1 (top blot) and PKCβII (middle blot) was detected by anti-V5 and anti-PKCβII, respectively. The blot was stripped and reprobed with antitubulin showing similar expression in all cases. D, E, Proteolytic enzymes (D) and organelles (E) are not involved in PKCβII-induced downregulation of TRPV1. HEK-293 cells were transfected with TRPV1–V5 or together with PKCβII, as indicated. Transfected cells were pretreated with Z-VAD (50 μm; to inhibit caspase), calpeptin (5 μm; to inhibit calpain), phenanthroline (Phe, 2 mm; to inhibit metallo-protease), MG132 (1 μm), and lactacystin (5 μm; to inhibit proteasome) for 20 h before solubilization. TRPV1 was detected by anti-V5 (top blot) followed by stripping and reprobing with antitubulin (bottom blot). F, Downregulation of TRPV1 by PKCβII was prevented by TRPV1 blockers. Tet-On HEK-293 cells were cotransfected with TRPV1–V5 and PKCβII-pTRE2. PKCβII was induced 24 h after transfection for an additional 24 h with (+) or without (−) ruthenium red RR (10 μm) or BCTC (10 μm), as indicated. The blot was probed for TRPV1 with anti-V5 (top blot), followed by stripping and reprobing with anti-PKCβII. The blot was then further stripped and reprobed with antitubulin to show equal sample loading in all cases (bottom blot). G, Downregulation of PKCβII by TRPV1 was prevented by RR. HEK 293 cells expressing TRPV1–V5 and PKCβII were treated with RR (10 μm) as indicated. The top blot shows the detection of PKCβII. The blot was stripped and reprobed with antitubulin (bottom blot). H, TRPV1 F641L protein expressed from HEK293 cells was probed with anti-V5 (top blot), followed by stripping and reprobing with antitubulin (bottom blot). All blots were repeated at least three times, with similar results. Molecular weights are shown on the right for all blots.