GATA3 phosphorylation regulates protein stability. A) T47D cells were treated or not with MPA and the PKA inhibitor PKI (1 μM) for eight hours. Nuclei were stained with propidium iodide and confocal microscopy analysis was performed. Scale bar = 10 μm. Right panel: Quantification of pSer308-GATA3 positive cells for the treatments performed in A. Total pSer308-GATA3 intensity relative to cell number was determined for a minimum of 150 cells per treatment (N = 6) (**P <0.01, ***P <0.001, one-way ANOVA). B) PKA induces pSer308-GATA3 in vitro. T47D cells treated as indicated were lysed and PKAs were immunoprecipitated from protein extracts from each cell treatment. GATA3 was immunoprecipitated from T47D cells growing in (D)MEM. Immunoprecipitated GATA3 was subjected to a cold in vitro phoshorylation assay as described in the Methods section. C) Confocal microscopy images of pSer308-GATA3 of cells in different stages of the cell cycle as determined by propidium iodide staining. Scale bar = 10 μm. The bar plot on the right panel represents a flow cytometry analysis of pSer308-GATA3 mean fluorescence intensity (MFI) of different stages of the cell cycle as determined by DNA content (***P <0.001, Student’s t-test). D) T47D cells were treated with CHX and/or with MPA or H89 (1 μM) for the indicated times. GATA3 bands of WBs underwent densitometry and values were normalized to GAPDH protein bands setting the value of untreated cells as 1.0. The results are graphically represented in a line plot. E) Cells were transfected with the indicated vectors for 48 hours, starved for 24 hours and treated as indicated. Densitometric analysis of WB was performed as in D. F) Cells were transfected as in E and densitometric analysis was performed as in D. G) Proposed model for GATA3 post-translational regulation by progestin. ANOVA, analysis of variance; CHX, cycloheximide; MPA, medroxyprogesterone acetate; PKA, protein kinase; WB, Western blot.