Journal:

Article Title: A requirement for sustained ERK signaling during thymocyte positive selection in vivo

doi: 10.1073/pnas.0505110102

Figure Lengend Snippet: Expression of Egr-1 and Id3 is biphasic in positively selected thymocytes and transient in negatively selected thymocytes. Egr-1 (A) or Id3 (B) expression in OT-I TAP° fetal thymic lobes incubated with OVAp, βCATp, or P815p for the indicated times. DP thymocytes were gated and analyzed for the expression of Egr-1 or Id3 by intracellular staining. The data are expressed as the MFI. Data are representative of at least three independent experiments.

Article Snippet: Cell lysates were electrophoresed in a 4-12% Bis-Tris gel, transferred to a polyvinylidene difluoride membrane, and probed for anti-mouse Id3 (Pharmingen) and ERK-2 (clone C-14) (Santa Cruz Biotechnology).

Techniques: Expressing, Incubation, Staining

Journal:

Article Title: A requirement for sustained ERK signaling during thymocyte positive selection in vivo

doi: 10.1073/pnas.0505110102

Figure Lengend Snippet: Id3 up-regulation does not depend on ERK activation or expression of Egr-1. (A) Expression of Id3 and phosphorylated ERK in OT-I TAP° fetal thymic lobes. The lobes were incubated with βCATp with or without the addition of 20 μM U0126 (MEK inhibitor), gated on DP thymocytes, and analyzed for induction of phosphorylated ERK at 2 min and expression of Id3 at 1 h. The MFIs of three lobes per time point were averaged, and the error bars represent the standard deviation. (B) Western blot of Id3 protein expression in OT-I TAP° thymocytes. Thymocytes were stimulated with OVAp-pulsed B6.SJL splenocytes with or without the addition of 20 μM U0126 for 3 h. DP thymocytes were separated by magnetic-activated cell sorting bead separation, and whole-cell lysates were prepared. Expression of Id3 was analyzed in Western blots by using an Id3 antibody. Blots were probed with anti-ERK2 antibodies to control equal loading of protein. The numbers below the blot indicate the fold induction of Id3 expression.

Article Snippet: Cell lysates were electrophoresed in a 4-12% Bis-Tris gel, transferred to a polyvinylidene difluoride membrane, and probed for anti-mouse Id3 (Pharmingen) and ERK-2 (clone C-14) (Santa Cruz Biotechnology).

Techniques: Activation Assay, Expressing, Incubation, Standard Deviation, Western Blot, FACS

Journal: Nature immunology

Article Title: Foxp3 and Toll-like receptor signaling balance T reg cell anabolic metabolism for suppression

doi: 10.1038/ni.3577

Figure Lengend Snippet: Glut1 expression increases the number and size of Treg cells but reduces their suppressive capacity. (a–c) Number (a), frequency (b) and size (by forward scatter (FSC)) (c) of Foxp3+ tTreg cells from the spleen of control and Glut1-tg mice, assessed by flow cytometry. (d) RNA-sequencing analysis of Treg cells from age-matched control and Glut1-tg mice (two independent cohorts), presented as normalized data. (e,f) Flow cytometry analyzing the expression of CD25, Foxp3, ID3 and EZH2 (e) or IFN-γ (f) by CD4+CD25− T cells isolated from the spleen of control and Glut1-tg mice and polarized under Treg cell–skewing conditions; results presented as the geometric mean fluorescent intensity (MFI) in e, right. Numbers adjacent to outlined areas (f) indicate percent IFN-γ+ cells. (g) Inhibition of Teff cell proliferation by control and Glut1-tg Treg cells as in e,f (presented as in Fig. 2e). *P < 0.05 (two-tailed Student’s t test). Data are representative of five independent experiments (a,b; mean + s.d.), four independent experiments (c; mean + s.d.), an experiment with two independent cohorts (two control mice with three Glut1-tg mice, and three control mice with one Glut1-tg mouse) (d), an experiment with four biological replicates per genotype (e,f; mean + s.d. in e) or three independent experiments (g).

Article Snippet: Antibodies used included anti-CD4 eFluor450 (eBioscience 48-0041-82), anti-CD25 PE (BD Biosciences 553866), anti-CD62L PE (eBioscience 12-0621-82), anti-CD69 PE (BD Biosciences 553237), anti-EZH2 PE (BD 562478), anti-FoxP3-APC (eBioscience 17-5773-80), anti-FoxP3-PE (eBioscience 12-5773-80), anti-Helios PE (eBioscience 12-9883-41), anti-ICOS PE (eBioscience 12-9942-81), anti-ID3 PE (BD Biosciences 564564), anti-mouse IFN gamma APC (eBioscience 17-7311-81), anti-mouse IL2 PE (BD Bioscience 561061), anti-KI67 eFluor450 (eBioscience 48-5698-80).

Techniques: Expressing, Flow Cytometry, RNA Sequencing Assay, Isolation, Inhibition, Two Tailed Test