ser9 5b3 cell signaling technology 9323p rrid ab 2115201 rabbit  (Cell Signaling Technology Inc)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Induction of IL-4 expression in CD4(+) T cells by thymic stromal lymphopoietin.

doi: 10.4049/jimmunol.178.3.1396

Figure Lengend Snippet: FIGURE 3. TSLP-mediated Il-4 transcript is partially Stat6 dependent. A, Splenic CD62LhighCD4 T cells from BALB/c mice were cultured with exogenous IL-2 (100 U/ml) and either TSLP (10 ng/ml) or IL-4 (10 ng/ml). Cells were cultured in the presence of plate-bound anti-CD3 mAb stimulation and either anti-IFN- mAb or anti-IL-4 mAb for the first 2 days. On day 5, cells were restimulated with plate-bound anti-CD3 mAb for 5 h in the presence of monensin, and IL-4 and IFN- expressions were measured by intracellular staining. Numbers in quadrants indicate the percentage of cells in the designated gate. Three independent experiments with identical results were performed. B, Splenic CD62Lhigh CD4 T cells from Stat6/ mice were cultured as described in A for 5 days. After restimulation with plate-bound anti-CD3 mAb and monensin, cytokine production was measured by intracellular staining. Numbers in quadrants indicate the percentage of cells in the designated gate. Three independent experiments with identical results were performed. C, D, and F, Splenic total CD4 T cells from BALB/c or Stat6/ mice were stimulated with either exogenous TSLP (10 ng/ml) or IL-4 (10 ng/ml) in the presence of plate-bound anti-CD3 mAb stimulation. The cells were harvested at each time point (0, 2.5, 5, 10, 20, and 30 h (C); 0, 2.5, 5, and 10 h (D and F)), and Il-4 or Il-4ra (Il-4r -chain) mRNA levels were measured by quantitative RT-PCR. All data were normalized to the cyclophilin B mRNA levels. Changes in mRNA expression are depicted as “relative change” to mRNA expression at time point 0. Three independent experiments with identical results for C and D and two independent experiments with identical results for F were performed. E, Splenic CD62Lhigh CD4 T cells from BALB/c mice were cultured without any cytokines or with either or both TSLP (30 ng/ml) and IL-4 (1 ng/ml) for 2 days in the presence of plate-bound anti-CD3 mAb stimulation. The cells were rested in culture medium without any cytokines for 8 h, recultured with the same cytokine for 10 min, and then lysed in radioimmunoprecipitation assay buffer for immunoblotting. Blots were probed with Abs specific for phospho-Stat6 (Y641) or total Stat6, as indicated. Two independent experiments with identical results were performed.

Article Snippet: Immunoblotting was performed with antiGata3 mouse mAb (clone HG3-31; Santa Cruz Biotechnology), anti-c-Maf rabbit polyclonal Ab (M-153; Santa Cruz Biotechnology), anti-Stat6 rabbit polyclonal Ab (Cell Signaling Technology), anti-phospho-Stat6 (Tyr641) rabbit polyclonal Ab (Cell Signaling Technology), and rabbit antitranscription factor IIB (TFIIB) polyclonal Ab (clone C-18; Santa Cruz Biotechnology).

Techniques: Cell Culture, Staining, Quantitative RT-PCR, Expressing, Radio Immunoprecipitation, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Induction of IL-4 expression in CD4(+) T cells by thymic stromal lymphopoietin.

doi: 10.4049/jimmunol.178.3.1396

Figure Lengend Snippet: FIGURE 5. TSLP-mediated induction of Gata3 is both Stat6 and IL-4 dependent. Splenic total CD4 T cells from BALB/c, Stat6/ (A), or Il-4/

Article Snippet: Immunoblotting was performed with antiGata3 mouse mAb (clone HG3-31; Santa Cruz Biotechnology), anti-c-Maf rabbit polyclonal Ab (M-153; Santa Cruz Biotechnology), anti-Stat6 rabbit polyclonal Ab (Cell Signaling Technology), anti-phospho-Stat6 (Tyr641) rabbit polyclonal Ab (Cell Signaling Technology), and rabbit antitranscription factor IIB (TFIIB) polyclonal Ab (clone C-18; Santa Cruz Biotechnology).

Techniques: