Fig. 4
PKA II activation and localization at the Golgi/TGN are required for efficient TSH-dependent CREB phosphorylation a Subcellular localization of PKA subunits in primary mouse thyroid cells. The catalytic (Cα) and regulatory subunits (RIα, RIIα and RIIβ) of PKA were visualized by immunofluorescence with specific antibodies. Nuclei were stained with DAPI. b, c Co-staining of RIIβ with antibodies against GOLPH4 (b) and TGN46 (c), showing selective localization of RIIβ subunit on membranes of Golgi and TGN, respectively. Left, Confocal images. Right, 3D reconstructions from confocal image stacks (see Supplementary Fig. 7). d Co-staining of RIIα with GOLPH4, showing preferential localization of RIIα at the Golgi. e Western blot analysis of CREB phosphorylation (Ser133) in response to TSH in cells pretreated for 1 h with a PKA I (Rp8-Br-cAMPS, 250 μM) or PKA II (Rp8-Br-PIP-cAMPS, 250 μM) inhibitor. α-tubulin was used as loading control. Data (mean ± S.E.M, n = 3) are expressed relative to those in the mock-stimulated sample pretreated with PKA I inhibitor. f Induction of early genes in response to TSH in cells pretreated for 1 h with PKA I/II inhibitors. Data (mean ± S.E.M, n = 4) are expressed as in e. g Effect of Ht31 or Ht31P (control) peptides (20 μM, 1 h) on the Golgi localization of RIIβ. Shown are representative RIIβ and GOLPH4 immunofluorescence stainings acquired with same contrast settings. h Quantification of images as in g. RIIβ intensity values (mean ± s.e.m., n = 12/20) are expressed relative to those of GOLPH4. i Western blot analysis of CREB phosphorylation (Ser133) in cells pretreated for 1 h with Ht31 or Ht31P (20 μM) and stimulated with TSH. Data (mean ± s.e.m., n = 3) are expressed relative to those measured in the mock-stimulated control sample. Differences in e, f and i are statistically significant by two-way ANOVA. *P < 0.05, **P < 0.01, by Bonferroni’s post hoc test. Difference in h is statistically significant by unpaired t-test (***P < 0.001). ns, statistically non-significant difference. Images in a–d and g are representative of 4, 3, 3, 5 and 3 independent experiments, respectively
