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. 2018 Jan 29;9:78. doi: 10.3389/fimmu.2018.00078

Figure 4.

Figure 4

Core fucosylation is required for the activation of CD4+ T cells. (A) Western blots analysis of T cell receptor (TCR). Whole cell lysates were resolved by SDS-PAGE on a 8% gel, transferred to a PVDF membrane, and probed with anti-TCRβ Ab. Densitometric analysis of the bands of TCRβ normalized against GAPDH. (B) Core fucose of N-glycan on TCRβ in Fut8−/−CD4+ T cells was detected by LCA blot. Whole cell lysates were immunoprecipitated with an anti-TCRαβ antibody. The immunoprecipitates were resolved by SDS-PAGE on a 8% gel, and probed with the LCA and anti-TCRβ Ab. (C) Histograms of binding capacity with LCA. Core fucosylation level on the surface proteins of Fut8+/+CD4+ T and Fut8−/−CD4+ T cells investigated by FACS analysis. (D) Downregulation of phosphorylated Zap70 in Fut8−/−CD4+ T cells. Purified CD4+ T cells were serum-starved and were stimulated with anti-CD3/CD28 Abs for 5 min at 37°C. Cells were lysated in lysis buffer for 15 min on ice. Whole cell lysates were subjected to 10% SDS-PAGE. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Densitometric analysis of the bands of pZAP70 normalized against ZAP70. Data are reported as the mean ± SD from three independent experiments (**p < 0.01; ***p < 0.001). (E) Loss of Fut8 reduced the CD4+CD69+ cells populations in the SPL after OVA immunization. SPLs were isolated from OVA-immunized and unimmunized mice (n = 5). Cells were stained with anti-CD69 and anti-CD4 Abs, and then detected by FACS. Data are reported as the mean ± SD from three independent experiments (*p < 0.05; ns, not significant). (F) Loss of Fut8 decreased the proliferation of CD4+ T cells. Purified CD4+ T cells were stimulated with anti-CD3ε Ab-coated microbeads and anti-CD28 Ab for 0, 6, 24, and 48 h at 37°C. The growth rates of CD4+ T cell were detected by MTT assay. Data are reported as the mean ± SD from three replicate cultures (*p < 0.05; **p < 0.01). The absorbance related to the formazan dye level was measured with a microplate reader at 570 nm.