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. 2021 Apr 19;12:658048. doi: 10.3389/fimmu.2021.658048

Figure 4.

Figure 4

SHARPIN is dispensable for activation, proliferation, survival, and differentiation of B cells stimulated with CD154. (A) Immunoblotting of phosphorylated p65 and total p65 protein levels in Sharpin+/+ and Sharpincpdm B cells after stimulation by CD154 for the indicated time. Representative of two independent experiments. (B) GSEA analysis of differentially expressed genes (DEGs) in Sharpin+/+ and Sharpincpdm B cells stimulated with CD154 for 24 h for the enrichment of p65 target genes (RNA-Seq data were pooled from two independent experiments). (C) Flow cytometry analysis of proliferation, survival, and CSR to IgG1 in Sharpin+/+ and Sharpincpdm B cells stimulated with CD154 or CD154 plus IL-4 for 96 h. (D) Flow cytometry analysis of survival, CSR to IgG1 and plasma cell differentiation in Sharpin+/+ and Sharpincpdm B cells after stimulation first with CD154 plus IL-4 for 48 h, washed, and then with CD154, alone or plus IL-4 or IL-21, for 48 h. (E) qRT-PCR analysis of gene expression, as indicated, in B cells stimulated with CD154, alone or plus IL-4, IFN-γ or IL-21, for 48 h. (F) The numbers of DEGs (p values < 0.01, absolute value of log2FC > 1) between Sharpin+/+ and Sharpincpdm B cells after stimulation with CD154 or CD154 plus IL-4 for 24 h, as depicted by Venn diagram. Data were pooled from two independent RNA-Seq experiments. * p<0.05; ** p<0.01; t-test.