Phosphorylation of GATA6 in HT-29 Cells

HT-29 cells were plated at 75,000 cells/cm², pretreated with human IFNγ for 24 hours (Roche), and then treated with TNF (100 ng/ml; PeproTech), insulin (500 ng/ml; Sigma), or both for 1 hour. Cells were lysed in NP-40 lysis buffer (51 (link)) supplemented with 10 mM sodium pyrophosphate and 30 mM sodium fluoride, and ~4 mg of cellular extract (adjusted according to total GATA6_L abundance based on immunoblotting) was incubated with 10 μl of phospho-GATA6_L (Ser³⁷) antiserum overnight on a nutator at 4°C. The following day, 30 μl of Protein A/G Plus UltraLink resin (Thermo) was added to the immune complexes for 1 hour on a nutator at 4°C. Beads were washed twice with ice-cold supplemented NP-40 lysis buffer and twice with ice-cold phosphate-buffered saline (PBS) before elution in Laemmli sample buffer (136 (link)). Samples were electrophoresed on a 10% polyacrylamide gel for 3 to 5 hours (until the 50-kD marker reached the bottom of the gel) before proceeding with quantitative immunoblotting as described (51 (link)). Densitometry was performed by integrating the pixel intensity (without lane-specific background subtraction) of the 75-kD band and, for the 60-kD form, the intensity between the 75-kD band and the upper shoulder of the immunoprecipitating antibody heavy chain (file S5).