S727A germ line mutation (STAT3 ^SA/SA ) does not alter T cell development under homeostatic conditions. (A) Recombinant catalytically active AKT was reacted with ATP and recombinant STAT3 protein. The phosphorylation of S727 was monitored by immunoblotting utilizing an antibody that recognizes p-STAT3 (S727). Densitometry was performed where p-STAT3 (S727) normalized to total STAT3 (N = 3). Mice with a S727A germ line mutation deletion does not alter homeostatic development. (B–D) Cells were isolated from the (B) thymus, (C) spleen, or (D) lymph nodes from mice with a S727A germ line mutation (STAT3^SA/SA) under homeostatic conditions. Cells were analyzed by flow cytometry for viability, CD3, CD4, CD44, or CD62L. Data represent mean ± SEM, n = 3. P values were calculated with a Student’s t test. (E) Primary naive murine CD4⁺ T cells isolated by negative selection (CD4⁺CD25⁻CD44^low) from wild type or STAT3 S727A STAT3^SA/SA mutant mice and cultured under Th17 conditions. Immunoblotting was performed on cell lysates for p-STAT3 (Y705), p-STAT3 (S727), and total STAT3. (E) Primary naive murine CD4⁺ T cells isolated by negative selection (CD4⁺CD25⁻CD44^low) from wild type or STAT3 S727A STAT3^SA/SA mutant mice and cultured under Th17 conditions for 4 days. (F–H) qPCR was utilized to measure the levels of (F) IL17a, (G) Rorc, and (H) cMYC transcripts. Source data are available for this figure: SourceData FS4.