Fig. 4

SUMO1 modification of TRPV1. a Conjugation of TRPV1 by GFP-SUMO1 co-expressed in CHO-K1 cells. Cells were transiently co-transfected with the following plasmids as indicated: Flag-TRPV1, GFP-SUMO1, RGS-SENP1, and RGS-SENP1m. Cell lysates were immunoprecipitated (IP) with anti-Flag antibody and analyzed by immunoblotting (IB) using anti-GFP or anti-Flag. Whole-cell lysates were also used for IB with anti-GFP, anti-Flag, and anti-RGS antibodies for input. Molecular weight standards (in kD) are shown on the left. b Similar to a for reciprocal IP using anti-GFP (for GFP-SUMO1) and IB by anti-Flag (for Flag-TRPV1). c Increased TRPV1 SUMOylation in DRG from the ipsilateral side of carrageenan-injected hindpaw of SENP1 cKO mice. Carrageenan (2%, 20 μl) was injected into the left hindpaw of SENP1 cKO and littermate control mice. L3–L4 DRG were dissected from the ipsilateral (Ipsi) and contralateral (Contra) sides 1 h after the injection. Lysates of DRG isolated from littermate control (Control) and SENP1 cKO (cKO) mice were subjected to IP with a control IgG or an anti-TRPV1 antibody, which were followed by IB using anti-SUMO1 and anti-TRPV1 antibodies. Note the higher level of SUMOylated TRPV1 in the Ipsi than the Contra side and the higher levels in SENP1 cKO than Control. Quantification of western blots in c were determined by measuring the relative intensity of TRPV1-SUMO band and the corresponding GAPDH band from the same lysate, which was then compared with Control performed in parallel in the same experiment. Data shown are means ± s.e.m. for three experiments. P = 0.0211, Control Contra vs. Control Ipsi; P = 0.0180, Control Contra vs. cKO Contra; and P = 0.0274 cKO Contra vs. cKO Ipsi by Student’s t test. d Potential SUMO modification sites in rat TRPV1 predicted by SUMOsp 2.0 software. e Loss of TRPV1 SUMOylation by K822R mutation. K324R and K822R substitutions in Flag-TRPV1 were made by site-directed mutagenesis and co-expressed with GFP-SUMO1 as indicated. Cell lysates were subjected to IP by anti-Flag, followed by IB for GFP and Flag. f Alignment of TRPV1 sequences from different species as indicated at the consensus SUMOylation site, with the conserved lysine in mammalian species shown in yellow. Note in chicken and alligator, the lysine is replaced by glutamate shown in blue. All blot images are representatives of three independent experiments