Activation of STAT-5 by treatment with SCFADs.

(A) Immunoblot analysis of total STAT-5 protein and phosphorylated STAT-5 protein in 32D cells. 32D cells were deprived of IL-3 for 18 hours, in the presence of various SCFADs, and then stimulated with 25 U/mL IL-3. Total cellular protein extracts harvested at 0, 5, 30, 60, and 120 minutes after IL-3 stimulation were treated with anti-Stat5 antibody, and the immunoprecipitated complexes were separated by SDS-PAGE and transferred to nitrocellulose. Filters were probed first with an antiphosphotyrosine, developed, stripped, and subsequently probed with an anti–STAT-5 antibody and developed. The figure is an autoradiogram of the ECL exposures. Treatments included Control (C); arginine butyrate at 1mM (AB); phenoxyacetic acid at 1mM (1); α methylhydrocinnamic acid at 1mM (2); 2,2 dimethyl butyric acid at 1mM (3); and 3-(3,4-dimethoxyphenyl) propionic acid at 1mM (4). (B) Densitometric quantitation of the films of the ECL immunoblots, expressed as arbitrary units, after subtraction of background and normalization for total Stat5 protein present in the sample. The dashed lines in all bar graphs indicate the level of phosphorylated STAT-5 protein remaining in the IL-3–only control at each specific time point. C, control; AB, arginine butyrate at 1mM; 1, phenoxyacetic acid at 1mM; 2, α-methyl hydrocinnamic acid at 1mM; 3, 2,2 dimethyl butyric acid at 1mM; and 4, 3-(3,4-dimethoxyphenyl) propionic acid at 1mM.