biotinylated  (Echelon Biosciences)

Journal: The Journal of Biological Chemistry

Article Title: Synergistic and additive interactions between receptor signaling networks drive the regulatory T cell versus T helper 17 cell fate choice

doi: 10.1016/j.jbc.2021.101330

Figure Lengend Snippet: Phosphatidylinositol metabolism is differentially regulated during Treg versus Th17 induction. A , a schematic of phosphatidylinositol metabolism is depicted. B , the label-free phosphoproteomic analysis determined the relative abundance of phosphopeptides for phosphatidylinositol kinases and phosphatases regulated by signaling input. C , primary CD4 + murine T cells isolated by negative selection were activated under Treg (anti-CD3 AB, soluble CD28 AB, and TGF-β) or Th17 (anti-CD3 AB, soluble anti-CD28 AB, TGF-β, and IL-6) induction conditions. Immunoblotting was performed for p-P85 (Y458) and total P85. Densitometry was performed across three biological replicates to quantitate p-P85 (Y458) normalized to total p85 levels. Shown are mean ± SD; p values were calculated by two-way ANOVA (∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01). D , primary murine CD4 + T cells were activated with under TH0 (anti-CD3, soluble anti-CD28 antibodies), Treg (anti-CD3 and soluble anti-CD28 antibody and TGF-β) and Th17 (anti-CD3 antibody, anti-CD28 antibody, TGF-β, and IL-6) for 10 min. P85 was immunoprecipitated. Immunoblotting was performed for P85 and P110. Densitometry was performed across three biological replicates. Each data point represents the mean ± standard deviation across three independent experiments and one-way ANOVA was performed. The abundances of ( E ) PtdIns(4,5)P2 and ( F ) PtdIns(3,4,5)P3 were measured with an imaging flow cytometry assay in primary murine CD4 + T cells activated under TH0, Treg, and Th17 conditions (defined above). Three biological replicates were performed. Presented is the mean ± standard deviation. G , primary murine CD4 + T cells were activated under Treg and Th17 conditions (defined above), and immunoblotting was performed on the resulting lysates for p-AKT(S473) and total AKT. Densitometry was performed on the immunoblots. Three biological replicates were included. Phosphorylated AKT was normalized to the total AKT abundance. Shown are mean ± SD; p values were calculated by two-way ANOVA (∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001). Source data are provided for the immunoblots in panels C , D , and G .

Article Snippet: Cells were also stained with antibodies against PtdIns(4,5)-PE (Echelon Biosciences Z-B045) or PtdIns(3,4,5)P3-PE (Echelon Biosciences Z-B3345B).

Techniques: Isolation, Selection, Western Blot, Immunoprecipitation, Standard Deviation, Imaging, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Synergistic and additive interactions between receptor signaling networks drive the regulatory T cell versus T helper 17 cell fate choice

doi: 10.1016/j.jbc.2021.101330

Figure Lengend Snippet: JAK2 inhibition dysregulates signaling networks during Th17 polarization. A , primary murine CD4 + T cells were activated for 10 min with constant amounts of anti-CD3 antibody, soluble anti-CD28 antibody, and TGF-β with varying amounts of IL-6. Immunoblotting was performed for p-ZAP7(Y319) and total ZAP70. Densitometry was performed on immunoblots across three biological replicates. Shown are mean ± SD. Primary murine CD4 + T cells were activated under Th17 (TCR+TGF-β+IL-6) or Treg polarization conditions (TCR+TGF-β) for 10 min in the presence or absence of 200 nM JAK2 inhibitor. B , immunoblotting was performed for p-ZAP7(Y319), ZAP70, p-CSK-S364, and CSK. Densitometry was performed on immunoblots across three biological replicates from panel B to determine the relative amount of ( C ) p-CSK-S364 and ( D ) p-ZAP70(Y319) normalized to total CSK and Zap70 abundance respectively. Shown are mean ± SD; p values were calculated by one-way ANOVA. E , primary murine CD4 + T cells were activated under Th17 (TCR+ TGF-β+IL-6) or Treg polarization conditions (TCR+ TGF-β) for 10 min in the presence or absence of 200 nM JAK2 inhibitor. Immunoblotting was performed for p-P85 (Y458) and P85. F , densitometry was performed on immunoblots across three biological replicates from panel E were p-P85(Y458) was normalized to total P85. Shown are mean ± SD; p values were calculated by one-way ANOVA. Mass ELISA assays were utilized to determine the relative level of ( G ) PtdIns(3,4,5)P3, and ( H ) PtdIns(4,5)P2 generated in primary murine CD4 + T cells activated under Th17 or Treg polarization conditions for 10 min in the presence or absence of the JAK2 inhibitor. Shown are mean ± SD from three biological replicates; p values were calculated by one-way ANOVA. I , primary murine CD4 + T cells were activated for 10 min with constant amounts of anti-CD3 antibody, anti-CD28 antibody, and TGF-β with varying amounts of IL-6. Immunoblotting was performed for p-AKT(S473) and total AKT. Densitometry was performed on immunoblots across three biological replicates. Shown are mean ± SD. Source data are provided for the immunoblots in panels A , B , E , and I .

Article Snippet: Cells were also stained with antibodies against PtdIns(4,5)-PE (Echelon Biosciences Z-B045) or PtdIns(3,4,5)P3-PE (Echelon Biosciences Z-B3345B).

Techniques: Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Generated

Journal: The Journal of Biological Chemistry

Article Title: Synergistic and additive interactions between receptor signaling networks drive the regulatory T cell versus T helper 17 cell fate choice

doi: 10.1016/j.jbc.2021.101330

Figure Lengend Snippet: Phosphatidylinositol metabolism is differentially regulated during Treg versus Th17 induction. A , a schematic of phosphatidylinositol metabolism is depicted. B , the label-free phosphoproteomic analysis determined the relative abundance of phosphopeptides for phosphatidylinositol kinases and phosphatases regulated by signaling input. C , primary CD4 + murine T cells isolated by negative selection were activated under Treg (anti-CD3 AB, soluble CD28 AB, and TGF-β) or Th17 (anti-CD3 AB, soluble anti-CD28 AB, TGF-β, and IL-6) induction conditions. Immunoblotting was performed for p-P85 (Y458) and total P85. Densitometry was performed across three biological replicates to quantitate p-P85 (Y458) normalized to total p85 levels. Shown are mean ± SD; p values were calculated by two-way ANOVA (∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01). D , primary murine CD4 + T cells were activated with under TH0 (anti-CD3, soluble anti-CD28 antibodies), Treg (anti-CD3 and soluble anti-CD28 antibody and TGF-β) and Th17 (anti-CD3 antibody, anti-CD28 antibody, TGF-β, and IL-6) for 10 min. P85 was immunoprecipitated. Immunoblotting was performed for P85 and P110. Densitometry was performed across three biological replicates. Each data point represents the mean ± standard deviation across three independent experiments and one-way ANOVA was performed. The abundances of ( E ) PtdIns(4,5)P2 and ( F ) PtdIns(3,4,5)P3 were measured with an imaging flow cytometry assay in primary murine CD4 + T cells activated under TH0, Treg, and Th17 conditions (defined above). Three biological replicates were performed. Presented is the mean ± standard deviation. G , primary murine CD4 + T cells were activated under Treg and Th17 conditions (defined above), and immunoblotting was performed on the resulting lysates for p-AKT(S473) and total AKT. Densitometry was performed on the immunoblots. Three biological replicates were included. Phosphorylated AKT was normalized to the total AKT abundance. Shown are mean ± SD; p values were calculated by two-way ANOVA (∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001). Source data are provided for the immunoblots in panels C , D , and G .

Article Snippet: Cells were also stained with antibodies against PtdIns(4,5)-PE (Echelon Biosciences Z-B045) or PtdIns(3,4,5)P3-PE (Echelon Biosciences Z-B3345B).

Techniques: Isolation, Selection, Western Blot, Immunoprecipitation, Standard Deviation, Imaging, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Synergistic and additive interactions between receptor signaling networks drive the regulatory T cell versus T helper 17 cell fate choice

doi: 10.1016/j.jbc.2021.101330

Figure Lengend Snippet: JAK2 inhibition dysregulates signaling networks during Th17 polarization. A , primary murine CD4 + T cells were activated for 10 min with constant amounts of anti-CD3 antibody, soluble anti-CD28 antibody, and TGF-β with varying amounts of IL-6. Immunoblotting was performed for p-ZAP7(Y319) and total ZAP70. Densitometry was performed on immunoblots across three biological replicates. Shown are mean ± SD. Primary murine CD4 + T cells were activated under Th17 (TCR+TGF-β+IL-6) or Treg polarization conditions (TCR+TGF-β) for 10 min in the presence or absence of 200 nM JAK2 inhibitor. B , immunoblotting was performed for p-ZAP7(Y319), ZAP70, p-CSK-S364, and CSK. Densitometry was performed on immunoblots across three biological replicates from panel B to determine the relative amount of ( C ) p-CSK-S364 and ( D ) p-ZAP70(Y319) normalized to total CSK and Zap70 abundance respectively. Shown are mean ± SD; p values were calculated by one-way ANOVA. E , primary murine CD4 + T cells were activated under Th17 (TCR+ TGF-β+IL-6) or Treg polarization conditions (TCR+ TGF-β) for 10 min in the presence or absence of 200 nM JAK2 inhibitor. Immunoblotting was performed for p-P85 (Y458) and P85. F , densitometry was performed on immunoblots across three biological replicates from panel E were p-P85(Y458) was normalized to total P85. Shown are mean ± SD; p values were calculated by one-way ANOVA. Mass ELISA assays were utilized to determine the relative level of ( G ) PtdIns(3,4,5)P3, and ( H ) PtdIns(4,5)P2 generated in primary murine CD4 + T cells activated under Th17 or Treg polarization conditions for 10 min in the presence or absence of the JAK2 inhibitor. Shown are mean ± SD from three biological replicates; p values were calculated by one-way ANOVA. I , primary murine CD4 + T cells were activated for 10 min with constant amounts of anti-CD3 antibody, anti-CD28 antibody, and TGF-β with varying amounts of IL-6. Immunoblotting was performed for p-AKT(S473) and total AKT. Densitometry was performed on immunoblots across three biological replicates. Shown are mean ± SD. Source data are provided for the immunoblots in panels A , B , E , and I .

Article Snippet: Cells were also stained with antibodies against PtdIns(4,5)-PE (Echelon Biosciences Z-B045) or PtdIns(3,4,5)P3-PE (Echelon Biosciences Z-B3345B).

Techniques: Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Generated

Journal: American Journal of Physiology - Cell Physiology

Article Title: Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain

doi: 10.1152/ajpcell.00305.2017

Figure Lengend Snippet: Primary antibodies used in this study

Article Snippet: PIP 3 Z-P345b Floating 1:100 Mouse Echelon Biosciences PIP 2 Z-B045 Floating 1:100 Mouse Echelon Biosciences Pals1 17710-1-AP Paraffin–TEG 1:100 Rabbit Proteintech Crumbs 3 crumbs 3A Paraffin–TEG 1:200 Rat Massey-Harroche, Le Bivic PKCζ sc216 Paraffin–TEG 1:500 Rabbit Santa Cruz Biotech.

Techniques: