Journal: The Journal of Experimental Medicine

Article Title: iNKT cell development is orchestrated by different branches of TGF-β signaling

doi: 10.1084/jem.20090127

Figure Lengend Snippet: Role of Tif-1γ/Smad4-independent signaling in the control of iNKT maturation. (A) tgf-b receptor expression analysis by RT-PCR performed on purified CD1d-αGalCer thymocytes from 16-d-old C57BL/6 mice. Relative gene expression are normalized on both hprt and g3pdh expression. (B) Flow cytometric analysis of CD1d-αGalCer tetramer + cells from iNKT cell-enriched thymocytes of C57BL/6 mice, stained for TGF-βRII. Means of fluorescence intensity (MFI) are reported with their SD. (C) jun-b , p21 , pme-pai expression analysis by RT-PCR performed on purified CD1d-αGalCer thymocytes from C57BL/6 mice. Relative gene expression was normalized on βactin expression. The results are representative of three different experiments from five to eight mice. (D) Flow cytometric analysis of the presence of iNKT cells in thymuses from either 13–14-d-old CD4-Cre x Tif-1γ fl/fl x Smad4 fl/fl mice or wild-type littermate mice and staining for CD44 and NK1.1 on CD1d-αGalCer tetramer + cells from the same thymuses. Absolute numbers of CD1d-αGalCer tetramer + cells among HSA low cells are illustrated by graphs, and absolute numbers for each development stage are illustrated in Table S1. The results are representative of three different experiments with at least three mice per group.

Article Snippet: The primers used are as follows: hprt , 5′-TCATTATGCCGAGGATTTGGA-3′ and 5′-CAGAGGGCCACAATGTGATG-3′; g3pdh , 5′-GCATGGCCTTCCGTGTCC-3′ and 5′-TGTCATCATACTTGGCAGGTTTCT-3′; tgfβRI , 5′-CAGACGAAGCAGACTGGACCAG-3′ and 5′-TGCTGCAATCAGGACCACTGC-3′; tgfβRII , 5′-GAAGAATACACCACCAGCAGTC-3′ ανδ 5′-ATGATGACAGCTATGGCAATCC-3′; p38 , 5′-ACCTAAAGCCCAGCAACCTAGC-3′ and 5′-GGTAGCCACGTAGCCTGTCATC-3′; mek1 , 5′-AACTGGGAGCTGGCAACGG-3′ and 5′-TGCGGGTTTGATCTCCAGGTG-3′; erk , 5′-GCTGACTCCAAAGCTCTGGATTTAC-3′ and 5′-CTCCTTAGGTAAGTCGTCCAACTCC-3′; p21 , 5′-TTGCACTCTGGTGTCTGAGC-3′ and 5′-GGGCACTTCAGGGTTTTCTC-3′; junB , 5′-GACGACCTGCACAAGATGAA-3′ and 5′-TGCTGAGGTTGGTGTAGACG-3′; pmepai , 5′-CAGGAGGAGAGACGATGGAC-3′ and 5′-AGGTAGGGGTAGGTGGGTTG-3′; and βactin SuperArray commercial Q-PCR primers.

Techniques: Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Purification, Gene Expression, Staining, Fluorescence