Fig. 10

Crosstalk between SUMOylation and PKC phosphorylation of TRPV1. a CHO-K1 cells were transfected with Flag-TRPV1 and its mutants together or not with GFP-SUMO1 or RGS-SENP1 as indicated. Cell lysates were subjected to IP by anti-Flag, followed by IB for GFP, Flag, and RGS. b CHO-K1 cells transfected with Flag-TRPV1, GFP-SUMO1, and RGS-SENP1 were untreated or treated with PMA (1 μM, 15 min) as indicated. Cell lysates were subjected to IP by anti-Flag, followed by IB for GFP, Flag, and RGS. c CHO-K1 cells transfected with Flag-TRPV1 and Flag-TRPV1-K822R were untreated or treated with PMA as indicated. To enhance the sensitivity, TRPV1 was enriched by IP using the anti-Flag antibody before IB was performed using the anti-phospho-S800 TRPV1 antibody (p-S800). ***P < 0.0001 for TRPV1 vs. TRPV1 K822R; TRPV1 vs. TRPV1+PMA; and TRPV1+PMA vs. TRPV1 K822R+PMA. d CHO-K1 cells transfected with Flag-TRPV1, GFP-SUMO1, and RGS-SENP1 were untreated or treated with PMA as indicated. The levels of phospho-S800 TRPV1 were determined as in c. Quantifications (means ± s.e.m.) of TRPV1 SUMOylation based on the results of IP Flag/IB GFP (a, b) and S800 phosphorylation based on the results of IP Flag/IB p-S800 (c, d) from three independent experiments are shown below the representative blots. e SUMOylation, but not PKC phosphorylation, at S502/S800 of TRPV1 is essential for inflammatory thermal hyperalgesia. Similar to Fig. 9f, but the left L3–L4 DRG of TRPV1^−/− mice were injected with AAV-TRPV1-S502/800A (n = 6) or AAV-TRPV1-S502/800A/K822R (n = 6). The mice were subject to the carrageenan edema model at 4 weeks after the injection. The paw withdrawal latency was measured by Hargreaves test. AAV-TRPV1-S502/800A, but not AAV-TRPV1-S502/800A/K822R, rescued thermal hyperalgesia. ***P < 0.0001 for AAV-TRPV1-S502/800A vs. AAV-TRPV1-S502/800A/K822R. Data are means ± s.e.m. Student’s t test for c and d, two-way ANOVA for e