Fig. 5: YAP/TAZ regulate chemokine expression prior to LTβR engagement.
From: YAP/TAZ direct commitment and maturation of lymph node fibroblastic reticular cells
a Immunoblot analyses at indicated time points and comparison of normalized pYAP/YAP ratio at 240 min in cultured FRCs derived from WT mice after stimulation with LTβR agonistic antibody (500 ng/ml) for indicated time points. b Immunoblot analyses of indicated proteins in nuclear (LaminB) and cytoplasmic (GAPDH) fractions of cultured FRCs after treatment with or without LTβR agonistic antibody. c Immunoprecipitation (IP) with anti-IgG or anti-YAP/TAZ (αY/T) antibody in primary cultured FRCs derived immunoblot with indicated antibodies. d Pull-down assay with streptavidin resin in HEK-293T cells after transfection with the streptavidin-binding peptide (SBP)-TAZ4SA, with or without plasmids encoding p52 or RelB and immunoblot analysis with indicated antibodies. e Pull-down assay with streptavidin resin in HEK-293T cells after transfection with the (SBP)-TAZ4SA, with or without plasmids encoding p52 (WT) or p52-Y293A mutants (YA) and immunoblot analysis with indicated antibodies. f Pull-down assay with streptavidin resin in HEK-293T cells after transfection with (SBP)-TAZ4SA or (SBP)-WW domain-deleted TAZ mutant (△WW) with or without plasmids encoding p52 or RelB and immunoblot analysis with indicated antibodies. g Diagram depicting the p52/RelB binding site within the mouse Ccl19 promoter and Ccl19 promoter-driven luciferase constructs containing p52/RelB binding site (WT) or the binding site deletion mutant (Mut). h Comparison of relative luciferase reporter activity using WT and Mut in HEK-293T cells. WT and Mut was co-transfected with or without p52 or p52 mutant (YA) and TAZ or TAZ mutant (△WW) in HEK-293T cells (n = 8). P values by one-way ANOVA. i Representative images of in situ proximity ligation assay showing localizations of YAP or TAZ and p52 after treatment with or without LTβR agonistic antibody in cultured FRCs. Nuclei are stained with DAPI. Scale bars, 50 µm. j ChIP experiments using IgG or anti-TAZ antibody were performed in MEFs infected with retrovirus encoding CTL or TAZ4SA with or without LTβR agonistic antibody. Unless otherwise denoted, similar findings were observed in three independent experiments. Horizontal bars indicate mean ± SD and P value versus 0 min or Control by two-tailed Student’s t-test. NS, not significant.
