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. 2020 Jun 22;130(8):3974–3986. doi: 10.1172/JCI126866

Figure 3. Expression of the 3 key regulators, Itgb1, Rps3, and Ywhaz, in T cells.

Figure 3

(A) Gene expression levels of the 3 regulators in undifferentiated and differentiated subtypes of CD4+ T helper cells isolated from mice. Th cell subsets were defined according to the expression pattern of transcription factors and/or cytokines: Tbx21 and IFN-γ for Th1, Gata3 for Th2, Rorc and IL-17 for Th17, and Foxp3 for Treg (see Methods). mRNA expression levels of the target genes were normalized to that of Gapdh (internal control). (B and C) Expression of Itgb1, Rps3, and Ywhaz proteins in the subtypes of CD4+ T helper cells determined by flow cytometry are presented as overlay histogram (B) and mean fluorescence intensity (MFI) (C). The differentiated Th cells were restimulated with PMA/ionomycin for 4 hours before intracellular cytokine staining. A gray-colored histogram indicates isotype control (Iso). Data are representative (B) or the mean ± SEM of 3 independent experiments (A and C). *P < 0.05, **P < 0.01, ***P < 0.001 as determined by 1-way ANOVA with a post hoc test (Tukey’s correction). (D) Western blot assay for Itgb1, Rps3, and Ywhaz in Th1 cells and Tregs. Data are representative of 3 independent experiments with similar results. (E) Western blot analysis showing coexpression of Ywhaz and Bcl2 in cell lysates or coexpression of Ywhaz and CD63 in exosomes isolated from Treg cells (n = 3).