Fig. 6: Heat stimulation stabilises TRPM3 in the DRG membrane.
From: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking
a The diagram shows the experimental procedure. The hindpaws of mice were soaked in a 43 °C water bath for 30 s, and the mice were placed at RT for 2, 5, 10, 20 or 40 min. Then, behavioural, co-IP and membrane protein immunoblotting experiments were performed. b, c ATF4/TRPM3 interactions (b) and TRPM3 membrane expression (c) in mice were evaluated at different time points after heat stimulation. The experiment was repeated four times in b. n = 6 mice per group in c. F(5,18) = 20.17, P = 0.0007 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0044 in 20 min in b. F(5,30) = 10.59, P = 0.0277 in 2 min, P = 0.0022 in 5 min, P < 0.0001 in 10 min, P = 0.0027 in 20 min in c. *P < 0.05, **P < 0.01 versus the RT group. d The diagram shows the experimental procedure. Isolated DRG neurons were placed in a 43 °C water bath for 30 s and placed at RT for 2, 5, 10, 20 or 40 min. The neurons were then lysed to detect the membrane abundance of TRPM3. e TRPM3 membrane expression in isolated DRG neurons was evaluated at different time points after direct heat stimulation. n = 3. F(5,12) = 12.3, P = 0.0358 in 2 min, P = 0.0100 in 5 min, P < 0.0001 in 10 min, P = 0.0085 in 20 min. *P < 0.05, **P < 0.01 versus the RT group. f–h Naïve (f), ATF4 siRNA-injected (g) and Atf4+/− (h) mice were subjected to the Hargreaves test at different time points after heat stimulation. n = 12 mice per group in f, g. n = 6 mice per group in h. F(5,66) = 14.36, P = 0.0004 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0057 in 20 min. **P < 0.01 versus RT group. One-way ANOVA followed by Tukey’s multiple comparisons test. The error bars indicate the SEMs.
