Fig. 4

Nufip2 protein is stabilized by Roquin. a Immunoblot analysis of Roquin, Nufip2, and Fmrp proteins in MEF lysates and supernatants or precipitates of b-isox-treated extracts. b Flow cytometry imaging analysis of GFP-Nufip2 localization in activated Th1 cells from Rc3h1^fl/fl; Rc3h2^fl/fl; Cd4-Cre-ERT2; rtTA mice. Before analysis, retrovirally transduced cells were treated with doxycycline for 24 h to induce GFP-NUFIP2 expression. c–f Immunoblot analysis of Nufip2 (c) or Nufip2 and Roquin (d–f) in c different mouse tissues, d mouse CD4⁺ T cells left untreated or stimulated for 12 h with (α-CD3) alone or in combination with anti-CD28 (α-CD28) or anti-ICOS (α-ICOS), e mouse CD4⁺ T cells with (Rc3h1/2^fl/fl) or without (Rc3h1/2^fl/fl; Cd4-Cre) Roquin expression, or f in lysates from wild-type and Roquin–deficient MEF cells, reconstituted with wild-type or Roquin-1 mutants as indicated. Gapdh (c–e) or α-tubulin (f) served as loading controls. g qPCR analysis of Nufip2 mRNA expression in cells from f. Expression was calculated relative to the reference gene Ywhaz and normalized to untransduced cells. Error bars in g represent mean and SD of two independent experiments. In a, b, d, e and f representatives of two (d, e, f) or three (a, b) independent experiments are shown