Figure 1. PLC activation increases TRPV1:AKAP150 association in TG neurons. A, Cultured TG neurons (24 well plates) were maintained in normal media and treated with Veh (0.1% EtOH), BK (1 μm), or m-3m3FBS (25 μm) for 5 min at 37°C, upon which total IP accumulation was measured. Data are illustrated as the mean number of disintegrations per minute ± SEM (n = 16 wells/treatment). B, Confocal immunocytochemistry of cultured TG neurons following treatment for 5 min at 37°C with Veh (0.1% EtOH), m-3m3FBS (m-3m3, 25 μm), or the PLC inhibitor U73122 (U7, 10 μm) and m-3m3FBS. Arrowheads denote plasma membrane expression of AKAP150 (green) in TRPV1 (blue)-positive neurons. Results are representative of 10 separate images taken from 5 individual coverslips. Scale bar, 50 μm. C, Coimmunoprecipitates and crude plasma membrane homogenate samples of cultured TG neurons, serum starved for 18 h, analyzed by Western blot following treatment for 5 min at 37°C with Veh (0.1% EtOH), m-3m3FBS (25 μm), or BK (1 μm). Results are representative of four independent trials. D, Densitometric quantification of Western blot data shown in C, data reported as mean ± SEM, n = 4. E, Coimmunoprecipitates of cultured TG neurons, serum starved for 18 h, analyzed by Western blot following treatment for 5 min at 37°C with Veh (0.1% EtOH), m-3m3FBS (25 μm), and/or GFX (10 μm). Results are representative of three independent trials. F, Densitometric quantification of Western blot data shown in E; data are reported as mean ± SEM, n = 3. Significance (compared with Veh treated) determined by one-way ANOVA with Bonferroni correction: *p < 0.05, **p < 0.01, ***p < 0.001. G, PKC activity of cleared TG culture lysates following treatment with Veh (0.1% EtOH, 5 min), PDBu (1 μm, 5 min), m-3m3FBS (25 μm, 5 min), BK (1 μm, 5 min), GFX (10 μm, 5 min pretreat)/PDBu (1 μm, 5 min), or GFX (10 μm, 5 min pretreatment)/m-3m3FBS (25 μm, 5 min). All cultures not pretreated with GFX received vehicle (0.1% DMSO) for 5 min before indicated treatment. Data reported as mean ± SEM, n = 6/treatment. Significance (compared with Veh treated, indicated with black asterisks, and compared with PDBu treated, indicated with white asterisks) was determined by one-way ANOVA with Bonferroni correction: **p < 0.01, ***p < 0.001. NS, No significance.