Fig. 7. Phosphorylation and destabilization of endogenous GATA6_L at 60 kD.

(A) Knockdown (shGATA6) and FLAG-tagged addback of GATA6_L at ~60 kD (red). Samples were immunoblotted for total (modified and unmodified) GATA6 (upper), FLAG (lower), and the indicated loading controls. (B) Destabilization of the 60-kD form of WT GATA6_L compared to the S37A-mutant addback cells. (C and D) Endogenous phospho-GATA6_L (Ser³⁷) immunoreactivity is not detectably affected by stimulation with TNF for 1 hour, inhibition with 20 μM CT99021 for 6 hours, or both. (E and F) Phosphorylation and destabilization of the 60-kD form of GATA6_L upon serum starvation. Specificity was confirmed by preincubation of cells with 20 μM CT99021 for 1 hour before serum starvation. (G and H) Phospho-GATA6_L (Ser³⁷) immunoprecipitation and total GATA6 immunoblot of HT-29 cells pretreated with IFNγ and stimulated with TNF ± insulin for 1 hour. The gamma of the immunoprecipitation image is set to 1.5 to minimize background from the immunoprecipitating antibody heavy chain. Input (0.5% input%) of each immunoprecipitate was immunoblotted for total GATA6 and the indicated loading controls. See file S5 for details. Data are means ± SEM of n = 3 (B, E, F, and H) or 6 (C and D) biological replicates.