Fig 1: DRAK2 inhibits JAK-1-mediated STAT5A phosphorylationEqual amounts of recombinant JAK1, STAT5A, and DRAK2 proteins were incubated in kinase reaction buffer containing ATP for 30 min. Reactions were separated by SDS-PAGE and analyzed by LC-MS/MS targeting the STAT5A Y694 residue.(A) For quantitative analysis, stable isotope-labeled peptides containing the unmodified and phosphorylated STAT5A Y694 peptides were spiked in to serve as internal standard controls. The absolute abundance of STAT5A Y694p is calculated as the MS1 peak area across all peptide-spectrum matches (PSMs) relative to the internal standard control AQUA peptide. Error bars represent standard deviation of three technical replicates. Data were analyzed with one-way ANOVA for comparison with STAT5A phosphorylation in the presence of JAK1 only, followed by a Šidàk multiple testing correction.(B) Abundance of STAT5A S780p relative to unmodified S780 in untargeted LC-MS/MS. Data are representative of three independent experiments. ***p < 0.001.
Fig 2: DRAK2 negatively regulates IL-2 signaling in NOD thymocytesSingle-cell suspensions from thymii and lymph nodes of 5-week-old NOD and NOD. Drak2-/- mice were stimulated with IL-2 for 15 min at 37°C. Cells were fixed and analyzed by flow cytometry to detect phosphorylated-STAT5 Y694 (pSTAT5), Foxp3, CD25, CD4, and CD8.(A) Representative flow cytometry plots gated on CD4 SP, Foxp3lo Treg precursors (CD25negFoxp3lo). The percent pSTAT5+ of (B) CD4 SP Foxp3lo Treg precursors (C) CD4 SP CD25+ Treg precursors (CD25+Foxp3neg), (D) mature thymic Tregs (CD4+CD8-CD25+Foxp3+), and (E) lymph node Tregs (CD4+CD8-Foxp3+) is plotted. Data were analyzed using a two-way ANOVA with a Šidàk multiple testing correction.(F) CD25, CD122, and CD132 MFI is shown for precursor and mature Tregs from 4- to 6-week-old NOD and NOD. Drak2-/- thymii. Data were analyzed using multiple t tests with the Holm-Šidàk correction.(G) Thymocytes from 1-day-old NOD.Drak2+/ and NOD. Drak2-/- mice were incubated for 24 h with medium alone or with increasing concentrations of IL-2. The absolute number and percent Foxp3+ cells of viable, CD4+CD8- cells is shown for three to four mice per group. Data were analyzed using two-way ANOVA with a Sida ` k multiple testing correction.(H) Four-week-old NOD and NOD. Drak2-/- mice were given either PBS or IL-2/anti-IL-2 complexes, i.p., daily, for 3 days. Thymii were harvested 48 h after the final injection and analyzed by flow cytometry to determine the proportion and absolute number of thymic, CD4+CD8-Foxp3+ Tregs. Treg expansion was compared using a simple linear regression. All data represent two independent experiments. Error bars represent standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.
Supplier Page from Abcam for Recombinant Human STAT5a protein