Fig. 5: Knockdown of PDE4DIP mislocalizes PKA RIIα from the Golgi apparatus and promotes its degradation.

A The production of cAMP was not increased in PDE4DIP-KD cells, as determined by a cAMP assay. Forskolin was used as a positive control. NS not statistically significant. B Western blot analysis of PDE4D and PKA RIIα (RIIα) expression in control and PDE4DIP-KD NSCLC cells. C Immunofluorescence staining showing the colocalization of PKA RIIα (RIIα, red) and PDE4DIP (green) in NSCLC cells. Scale bars, 10 μm. D, E Coimmunoprecipitation of PDE4DIP and PKA RIIα in PDE4DIP-overexpressing cells. The PDE4DIP-Myc plasmid was transfected into H1299 and A549 cells. Immunoprecipitation was performed with either an anti-PKA RIIα (RIIα) or an anti-Myc (PDE4DIP) antibody. TCL total cell lysate. F Immunofluorescence analysis of PKA RIIα (red) and GM130 (green) colocalization at the Golgi apparatus in control (shNC) and PDE4DIP-KD (shP1) NSCLC cells. Scale bars, 10 μm. Representative images (left) and colocalization values (Rcoloc) of PKA RIIα and GM130 are shown (right). The average co-localization was calculated using the JACoP plug in of the ImageJ program in 10–15 images obtained for each condition. The data are presented as the means ± SD of three biological replicates, and the P values were determined by Student’s t test. ***P < 0.001 vs. control. G Western blot analysis of PKA RIIα ubiquitination in control and PDE4DIP-KD NSCLC cells.