Fig. 4
PKCθ is involved in the TAAR1-mediated IA response. a The expression of transcripts of novel PKC isozymes (θ, ε, δ and η) in mouse TGs. Samples without reverse transcriptase (-RT) were used as negative controls. b Summary data demonstrating the effect of tyramine (0.1 µM) on IA in small TG neurons dialyzed with PKCη-PSI (10 µM, n = 9 cells), PKCθ-IP (10 µM, n = 9 cells), δV1-1 (1 µM, n = 10 cells) or εV1-2 (2 µM, n = 11 cells). **p < 0.01 vs. control, paired t test. c Western blot analysis of PKCθ protein abundance in TG cells treated with NC-siRNA or PKCθ-siRNA. The immunoblots are representative of the results of at least three independent experiments. **p < 0.05 vs. NC-siRNA, unpaired t test. d Representative traces (left panel) and summary data (right panel) showing that PKCθ-siRNA treatment abrogated the 0.1 µM tyramine-induced IA response (n = 12 cells). Tyramine at 0.1 µM significantly decreased IA in NC-siRNA-treated TG neurons (n = 12 cells). *p < 0.05 vs. control + NC-siRNA, unpaired t test. e Immunofluorescence analysis of PKCθ translocation mediated by 0.1 µM tyramine. The white arrows indicate the line-scanned area. Data are representative of three separate experiments. f Western blot analysis of PKCθ expression in cytoplasmic and membrane fractions isolated from TG cells treated with 0.1 µM tyramine. α-Na+/K+ ATPase served as an indicator for membrane contamination of cytosolic extracts. β-actin was used as a control for protein loading. The immunoblots are representative of the results of at least three independent experiments. **p < 0.01 vs. control, unpaired t test