Journal: The Journal of Experimental Medicine

Article Title: Counter-regulation of T cell effector function by differentially activated p38

doi: 10.1084/jem.20131917

Figure Lengend Snippet: p38 Alternative activation is required for TCR-induced expression of NFATc1 and IRF4 in CD4 + T cells. (A) Purified CD4 + T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and immunoblotted for the indicated transcription factors 48 h later. (B and C) Purified CD4 + T cells were stimulated with anti-CD3/CD28 in the presence of 11R-VIVIT or 11R-VEET. Irf4 mRNA (B) and protein (C) were determined 24 h later. (D and E) Purified CD4 + T cells from WT and DKI mice were stimulated with anti-CD3/CD28 or PMA plus ionomycin, as indicated for the indicated times and analyzed for expression of Nfatc1 and Irf4 mRNA (D) and protein (E). Results shown in A–E are representative of three independent experiments each. (F) Naive CD4 + T cells were stimulated with either anti-CD3/CD28 or PMA and ionomycin under neutral or Th17-skewing conditions for 3 d. IL-17A and IFN-γ expression were determined by intracellular staining and flow cytometry after restimulation with PMA and ionomycin. Results shown in the left panel are representative of three independent experiments and bar graphs in the right panel show the mean ± SEM of all three. *, P < 0.05 (unpaired two-tailed Student’s t test).

Article Snippet: Anti–human phospho NFATc1 (S172; clone 679340) was purchased from R&D Systems.

Techniques: Activation Assay, Expressing, Purification, Staining, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Counter-regulation of T cell effector function by differentially activated p38

doi: 10.1084/jem.20131917

Figure Lengend Snippet: MAPK-cascade activated p38 inhibits functions distal to alternatively activated p38. (A) Purified CD4 + T cells were cultured overnight without stimulation, and then treated with either UV (50 J/m 2 or 100 J/m 2 or 200 J/m 2 ), 0.6 M sorbitol for 30 min, or PMA (10 ng/ml) and ionomycin (1 µg/ml) for 1 h. Cell lysates were immunoblotted with anti-phospho-p38. The results are representative of three independent experiments. (B) Purified CD4 + T cells were treated with either 0.6 M sorbitol for 30 min, 50 J/m 2 UV, or PMA and ionomycin were stimulated with anti-CD3/CD28 for 24 h. The expression of Nfatc1 and Irf4 was determined by quantitative real-time PCR. Results are the mean ± SEM of three independent experiments. *, P < 0.05 (unpaired two-tailed Student’s t test). (C) Cells were treated as in B, and IRF4 expression was determined by Western blot. The results are representative of two independent experiments. (D) Freshly purified T cells were stimulated or not with anti-CD3/CD28 for 48 h. Cytosolic and nuclear fractions were separated in a low percentage SDS-PAGE (8%) gel and immunoblotted for NFATc1. The results are representative of three independent experiments. (E) CD4 + T cells were stimulated for 48 h with anti-CD3/CD28, and then treated with sorbitol in the presence or absence of SB203580 and/or SP600125 for 30 min, rested for 1 h, then immunoblotted for NFATc1 in cytosolic and nuclear fractions. Results are representative of three independent experiments. (F) Purified CD4 + T cells were treated or not with sorbitol for 30 min and stimulated with anti-CD3/CD28 for 48 h, and then immunoblotted for NFATc1 in cytosolic and nuclear fractions. The results are representative of three independent experiments. (G) Jurkat T cells were stimulated for 48 h with an agonistic anti-TCR antibody (C305), and then treated with sorbitol in the presence or absence of SB203580 and/or SP600125 for 30 min. After 1 h without further stimulation, cytosolic fractions were immunoblotted for phospho-NFATc1 (p-NFATc1). The results are representative of three independent experiments. (H) Recombinant p38 was activated with Zap70, MKK6, or buffer alone in in vitro kinase buffer. After 1 h, recombinant ATF2 and 10 µCi [ 32 P]ATP were added for 30 min before separation on SDS-PAGE and PhosphorImager analysis. The phosphorylation state of p38 Y323 was determined by immunoblotting. The results are representative of three independent experiments. (I) Recombinant p38 was activated with Zap70, MKK6, or buffer alone and an in vitro kinase assay using recombinant NFATc1 as substrate was performed as in (H). The results are representative of two independent experiments. (J) Recombinant p38 was activated with MKK6 or buffer alone in in vitro kinase buffer. Active JNK1 was kept in in vitro kinase buffer. After 1 h, recombinant NFATc1 was added and incubated for an additional hour before separation on SDS-PAGE and immunoblotting with an antibody specific for NFATc1 phosphorylated on residue S172. The results are representative of two independent experiments. (K) Recombinant p38 was activated with Zap70, MKK6, or buffer alone in an in vitro kinase assay with recombinant NFATc1 as the p38 substrate, as in I. Samples were separated on a low percentage SDS-PAGE (8%) and immunoblotted for NFATc1 phosphorylated on residue S172. The results are representative of three independent experiments.

Article Snippet: Anti–human phospho NFATc1 (S172; clone 679340) was purchased from R&D Systems.

Techniques: Purification, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, SDS Page, Recombinant, In Vitro, Phospho-proteomics, Kinase Assay, Incubation, Residue

Journal: Chinese Journal of Cancer

Article Title: Expression and unique functions of four nuclear factor of activated T cells isoforms in non-small cell lung cancer

doi: 10.5732/cjc.010.10156

Figure Lengend Snippet: Expression of NFAT proteins and CaN in non–small cell lung cancer (immunohistochemistry). A, negative staining of NFAT1; B–E, positive staining of NFAT1, NFAT2, NFAT3, and NFAT4, respectively; F, positive staining of CaN. Bars , 100 µm.

Article Snippet: Mouse anti-human NFAT2 and mouse anti-human NFAT4 antibodies were purchased from Santa Cruz, Inc. Rabbit anti-human antibodies were purchased from Canadian Chemicom International, Inc. PV9000 and DAB reagents were purchased from Beijing Zhongshan Goldenbridge Biotechnology, Ltd.

Techniques: Expressing, Immunohistochemistry, Negative Staining, Staining