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. Author manuscript; available in PMC: 2015 Apr 29.
Published in final edited form as: Immunol Cell Biol. 2014 Jun 17;92(8):721–728. doi: 10.1038/icb.2014.43

Figure 1. Generation and characterization of Themis1 mutants lacking individual conserved domains.

Figure 1

a. Top, Schematic of Themis1 protein structure showing the location of the two novel globular CABIT domains (CABIT-1 and CABIT-2) each containing a conserved cysteine (Cys) residue, the bipartite nuclear localization signal (NLS), and the proline-rich region (PRR). Bottom, amino-acid sequences of relevant regions that are mutated or deleted in the three Themis1 variants used in this study (ΔPRR, ΔNLS and ΔCys). b. Association of Themis1 and Themis1 mutants with the adaptor Grb2. Flag epitope tagged wild-type (WT) Themis1 or Themis1 mutant proteins were overexpressed in HEK-293T cells. Themis1 proteins were immunoprecipitated with anti-FLAG-Sepharose beads, resolved by SDS-PAGE and blotted for Grb2. c. Subcellular localization of Grb2 in Themis1−/− and Themis1+/+ thymocytes. Cytoplasmic and nuclear fractions of thymocytes were immunobloted for Themis1, PLCγ-1(cytoplasmic control) and Lamin B (nuclear control). d. Subcellular localization of Themis1. Cytoplasmic and nuclear fractions of HEK-293T cells overexpressing WT or mutant Themis1 proteins were immunobloted for Themis1, PLCγ-1(cytoplasmic control) and Lamin B (nuclear control).