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

Figure 7.

Figure 7

SHARPIN inhibits caspase 8 and caspase 9 activation in B cells stimulated with CD154 and IL-21. (A) Immunoblotting analysis of STAT3 and STAT5 activation in Sharpin+/+ and Sharpincpdm B cells stimulated with CD154 for 24 h and then IL-21 for different periods of time, as indicated. (B, C) Immunoblotting analysis of BCL2, BCL-XL and BIM expression and caspase 3 activation (B) and cytochrome C released into the cytosol (C) in Sharpin+/+ and Sharpincpdm B cells stimulated for 48 h. (D) qRT-PCR analysis of gene expression, as indicated, in Sharpin+/+ and Sharpincpdm B cells stimulated with CD154, alone or with IL-4 or IL-21, for 24 h (*p<0.05; **p < 0.01; t-test). (E–F) Immunoblotting analysis of caspase 8 and caspase 9 activation, expression of cFLIPL, XIAP and BCL2 family members as well as BAD activation in Sharpin+/+ and Sharpincpdm B cells stimulated with CD154 and IL-21 for different periods of time, as indicated. Quantified signals of caspase 8 activation, cFLIPL cleavage and total cFLIPL levels were depicted in (F). (G) qRT-PCR analysis of Cflar expression in Sharpin+/+ and Sharpincpdm B cells stimulated with CD154 plus IL-21 for 24 h (t-test). (H) Proteins in whole cell lysates (input) and immunoprecipitated by anti-cFLIP were analyzed by immunoblotting for linear M1-Ub and K48 poly-Ub levels in Sharpin+/+ and Sharpincpdm B cells stimulated with CD154 plus IL-21 for 48 h. All immunoblotting analyses were representative of two independent experiments.