Extended Data Fig. 7: Molecular determinants of Roquin-1 interaction with Regnase-1.
From: Disrupting Roquin-1 interaction with Regnase-1 induces autoimmunity and enhances antitumor responses
(a) Lysates of WT or iKO CD4+ T cells were subjected to immunoprecipitation (IP) with antibodies against Regnase-1. Input lysates before IP and eluates from beads after IP were analyzed in immunoblots with antibodies against Roquin-1 or Regnase-1. (b, d) Fluorescence microscopy images of HeLa cells transfected with BFP-Rck and GFP-Regnase-1 (GFP-Reg-1) and stained with ER staining dye (b), or of cells transfected with GFP-Regnase-1aa112-297 and mCherry-Roquin-1aa1-510 (d). Quantification of GFP fluorescence lifetime in the cytoplasm (cyto) and nucleus (nuc) (e) of transfected cells shown in (d). (c) Cytoplasmic lysates of CD4+ T cells were fractionated after sucrose gradient centrifugation and distribution of Roquin, Regnase-1 and Rpl7a proteins in the individual fractions analyzed via immunoblots, as indicated. Above, representative absorbance profile obtained during fractionation of gradients with indication of localization of 40 S and 60 S ribosomal subunits, 80 S monosomes and polysomes. (f, g) SDS-PAGE of competitive in vitro GST-pulldown experiments using GST-regnase-1D141N and SUMO-roquin-1aa2-440 in combination with the indicated untagged Roquin-1aa2-440 double or single mutants. Purified proteins before pulldown (IN), supernatants of wash steps (W) as well as eluted proteins (E) were loaded. Asterisks mark the migration of degradation products of GST-regnase-1. (h) Quantification of eluted mutant Roquin-1aa2-440 relative to SUMO-roquin-1aa2-440 wild-type protein of in vitro GST-pulldown experiments depicted in (g). (b, d, f, g) Depicted are representative data of n = 3 independent experiments or (a, c) of n = 2 independent experiments. (e) Data are presented as mean + /- SEM or (h) mean + /-SD and (h) statistical significance was calculated using t tests.
