Dataset Information

TCR phosphoproteome using DP thymocytes stimulated by H2-Kb MHC tetramers

ABSTRACT: Quantitative phosphoproteome with OT-I TAP-/- mice DP thymocytes stimulated by H2-Kb tetramers for 2 min. The quantitative data were calculated by the spike-in standard lysates from DPK cells cultured in the heavy media containing 15N2 13C6-lysine and 15N4 13C6-arginine.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Keiichi Nakayama

PROVIDER: PXD036851 | JPOST Repository | Thu Jul 27 00:00:00 BST 2023

REPOSITORIES: jPOST

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Dataset's files

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			Action	DRS
	orb_160422_A-SF_Q4H7_%231.raw	Raw
	orb_160422_A-SF_Q4H7_%232.raw	Raw
	orb_160422_A-SF_Q4H7_%233.raw	Raw
	orb_160422_A-SF_Q4H7_%234.raw	Raw
	orb_160422_A-SF_Q4H7_%235.raw	Raw

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Project description:It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level TCR stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level TCR stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments as being differentially regulated by N-ras (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4+ T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types. Since we were interested primarily in genes that were differentially regulated by N-ras following a low-level TCR stimulus, our microarray data comparison was between data from TCR-stimulated, WT CD4+ T-cells and from TCR-stimulated, N-ras KO CD4+ T-cells. Genes that were differentially regulated in the comparison between stimulated N-ras KO CD4+ T-cells and unstimulated N-ras KO CD4+ T-cells, as well as those genes that were differentially regulated in the comparison between stimulated WT CD4+ T-cells and unstimulated WT CD4+ T-cells were excluded from this analysis. To determine if N-ras and H-ras regulate different sets of genes in thymocytes, a comparison was made between the set of genes that were differentially regulated by N-ras in the [WT] vs. [N-ras KO] comparison and the set of genes that were differentially regulated by H-ras in the [WT] vs. [H-ras KO] comparison. RNA was extracted from CD4+ T cell splenocytes isolated from 6-20 week old N-Ras KO and WT mice following growth in T cell growth media either with or without 1 microgram/milliliter ant-CD3 and anti-CD28 antibodies. RNA was extracted from thymocytes isolated directly from 6-20 week old N-Ras KO, H-Ras KO and WT mice.