Fig. 7
Interfering with retrograde trafficking or Golgi/TGN organization impairs cAMP/PKA signaling and CREB phosphorylation. a Sequential HILO images showing effect of BFA on TGN organization and TSH/TSHR retrograde trafficking. Primary mouse thyroid cells were transfected with the TGN maker ST-RFP. Thereafter, they were treated with BFA for 30 min, stimulated with TSH-647 for 10 min and imaged for additional 30 min. b Immunofluorescence against RIIβ and either GOLPH4 (left) or TGN46 (right) in cells pretreated with BFA, showing displacement of the PKA RIIβ subunit from the Golgi and its reduced association with the TGN, respectively. Images were acquired by confocal microscopy. Data in a and b are representative of 3 independent experiments. c cAMP FRET responses (mean ± s.e.m.; n = 9/13) in primary thyroid cells isolated from Epac1-camps mice pretreated with or without (control) BFA for 30 min. d PKA FRET responses (mean ± s.e.m.; n = 11/11) in primary thyroid cells transfected with the AKAR2 sensor and pretreated with or without (control) BFA. Data in c and d were normalized as in Fig. 5. Differences at individual time points were compared using the Holm–Šídák test for multiple comparisons (black dots, P values). e Western blot analysis of CREB (Ser133) phosphorylation in primary thyroid cells stimulated with TSH with/without BFA pretreatment. α-tubulin was used as loading control. Images are representative of 3 independent experiments. Data (mean ± s.e.m., n = 3) are expressed relative to those measured in the mock-stimulated control sample. f Western blot analysis of CREB (Ser133) phosphorylation in primary thyroid cells transfected with Vps35 siRNA pool or control non-targeting siRNA and stimulated with TSH for 10 min. α-tubulin was used as loading control. Images are representative of six independent experiments. Data (mean ± s.e.m., n = 6) are expressed relative to those measured in the mock-stimulated control sample. Differences in e and f are statistically significant by two-way ANOVA. *P < 0.05 by Bonferroni’s post hoc test. g Schematic view of the results
