pe anti human ps6 antibody  (Cell Signaling Technology Inc)

Journal: Science translational medicine

Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway.

doi: 10.1126/scitranslmed.aal3322

Figure Lengend Snippet: Fig. 6. Endothelial Jagged1 induces T effector cells via the Notch1-mTORC1 pathway. HMVECs were preconditioned with the indicated plasma, loaded with anti-CD3 antibody, and overlaid with CD4 T cells, as in Fig. 4A. Intracellular expression of pS6, T-bet, or RORgt was analyzed by flow cytometry. All data are means ± SEM. (A and B) pS6 expression in CD4 T cells was measured after 24 hours. Representative histograms and collective MFIs are from three to six independent experiments. (C and D) Effect of the Notch1 signaling inhibitor DAPT (10 mM) or vehicle on the induction of pS6. Data are from six experiments. (E to H) Effect of the mTORC1 inhibitor rapamycin (RAPA; 50 nM) or the AKT inhibitor VIII (AKT Inh; 5 mM) on the induction of T-bet+ and RORgt+ CD4 T cells quantified after 4 days. Data are from five to six experiments. (I) Healthy CD4 T cells were transfected with Raptor siRNA or control siRNA and analyzed for RPTOR mRNA expression 12 hours later by quantitative PCR (qPCR). Data are from five independent experiments. (J) CD4 T cells from healthy donors were transfected with Raptor siRNA or control siRNA, respectively, and cocultured with pretreated ECs, as described above. Data are from six experiments. (K and L) Expression of pS6 in CD4 T cells was measured in freshly isolated PBMCs from GCA patients and healthy donors. Representative histograms and data are from five control-patient pairs. (M to O) Expression of Notch1, pS6, T-bet, and RORgt in CD4 T cells was determined by multiparametric flow cytometry in freshly isolated PBMCs of GCA patients. Each dot represents an individual patient.

Article Snippet: Multiparametric flow cytometry panels were assembled with antibodies to Notch1, CD45RA, CD25, HES1, T-bet, RORgt, and pS6, combined with anti-CD3 and anti-CD4 antibodies as follows: APC-Cy7 (allophycocyanin-Cy7) anti-human CD3 antibody (clone HIT3a, BioLegend), FITC (fluorescein isothiocyanate) or PE-Cy7 (phycoerythrinCy7) anti-human CD4 antibody (clone RPA-T4, BioLegend), APC or APC-Cy7 anti-human CD45RA antibody (clone HI100, BioLegend), PE anti-humanCD25antibody (cloneM-A251, BioLegend),APCorPE antihuman Notch1 antibody (clone MHN1-519, BioLegend), purified antihuman HES1 antibody (clone 4H1HES, eBioscience) plus PE-Cy7 anti-mouse IgG1 antibody (clone M1-14D12, eBioscience), PE or PE-Cy7 anti-human T-bet antibody (clone eBio4B10, eBioscience), APC anti-human RORgt antibody (clone AFKJS-9, eBioscience), Parameters Patients with GCA Patients with RA Number of patients 80 54 Age (years) (mean ± SD) 69.63 ± 7.95 53.16 ± 16.35 Female 77.5% 79.63% Headaches 70% Eye involvement 31.25% Jaw claudication 26.25% Polymyalgia rheumatica 60% Aortic/large vessel involvement 43.75% Prednisone (mg/day) (mean ± SD) 7.49 ± 11.29 Disease duration (months) (mean ± SD) 14.36 ± 12.40 14.88 ± 11.48 ESR (mm/hour) (mean ± SD) 51.43 ± 35.39 24.63 ± 19.53 CRP (mg/dl) (mean ± SD) 6.25 ± 7.48 1.72 ± 4.47 Untreated patients 32.5% 16.67% 12 of 15 SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 and PE anti-human pS6 antibody (cloneD57.2.2E, Cell Signaling Technology).

Techniques: Clinical Proteomics, Expressing, Flow Cytometry, Transfection, Control, Real-time Polymerase Chain Reaction, Isolation