Fig. 6: CNR2 binds to JAK1 and inhibits the STATs signaling.

CNR2 binds to JAK1 and inhibits the STATs signaling. a, b Gene Ontology (GO) category analysis and Heatmap GO analysis of RNA-seq data of Cnr2^CKO and Cnr2^GFP CD8⁺ T cells treated with anti-CD3 plus (5 μg/ml) anti-CD28 (5 μg/ml) for 24 h by using Metascape website (http://metascape.org). c GSEA analysis of the differentially expressed genes (RNA-seq datasets) of the JAK-STAT signaling pathway in Cnr2-deficient CD8⁺ T cells versus wild-type CD8⁺ T cells. d qPCR validation of the expression of genes downstream JAK-STAT signaling pathway in Cnr2-deficient and wild-type CD8⁺ T cells (Left), and in THC-treated Cnr2-deficient and wild-type CD8⁺ T cells (Right). Data are presented as the mean ± SD. of three biological replicates. **P < 0.01. e Mass spectrum analysis of CNR2 associated proteins in CD8⁺ T cells from Cnr2-2xFlag-IRES-Egfp^flox/flox reporter mice after Flag pull-down assay. Six protein bands detected by silver-staining in the FLAG group but not in the IgG group were cut and performed mass spectrum analysis. f The top ten identified peptides were shown in the list. g Cnr2-2xFlag-IRES-Egfp^flox/flox CD8⁺ T cells were treated with THC or DMSO as a control for 24 h. Cell lysates were then immunoprecipitated with Flag antibody and analyzed by immunoblot with anti-JAK1 and anti-Flag. h Cnr2^GFP and Cnr2^CKO CD8⁺ T cells were stimulated with anti-CD3 plus anti-CD28 for 10–30 min. Cell lysates were analyzed by immunoblot with anti-phosphorylated JAK1, anti-phosphorylated STAT1, anti-phosphorylated STAT3, anti-total STAT1, and anti-total STAT3. i Cnr2^GFP and Cnr2^CKO CD8⁺ T cells were pretreated with THC or DMSO for 24 h and then stimulated by anti-CD3 plus anti-CD28 for 30 min. Cell lysates were analyzed by immunoblot with anti-phosphorylated JAK1, anti-phosphorylated STAT1, anti-phosphorylated STAT3, anti-total STAT1, and anti-total STAT3.