Generation of SHD1-deficient mice. (A) Structure of the targeting vector and the targeting strategy. The filled boxes represent exons. The targeting vector was designed to replace SpeI-NotI fragment containing the proximal promoter and the 5′-portion of exon 1 with neomycin resistance cassette. This removes all 5 potential translational start sites in exon 1. Neo indicates neomycin resistance cassette; DTA, diphtheria toxin A; S, SalI; B, BamHI; Sp, SpeI; N, NotI; and A, ApaI. (B) Southern blot analysis of BamHI-digested genomic DNA from wild-type (+/+), heterozygous (+/−), or homozygous (−/−) mice. The wild-type (8.0 kb) and the mutant (5.8 kb) bands are indicated by arrows. (C) The expression of SHD1 in mutant mice. Total RNA was extracted from splenocytes of mutant mice, and the expression of SHD1 was analyzed by RT-PCR. GAPDH is shown as a control. (D) The absence of SHD1 protein in SHD1-deficient mice. The nuclear extracts of thymocytes from the mutant mice were subjected to a Western blot analysis using anti-SHD1 antibody. α-Tubulin was probed to indicate equal loading of the protein extracts. (E) Association of SHD1 and STAT5 in wild-type (+/+) and mutant (−/−) mast cells. STAT5 was immunoprecipitated from the nuclear extracts with anti-STAT5 antibody or control IgG. The precipitated proteins were analyzed by SDS-PAGE and Western blotting using anti-SHD1 or anti-STAT5 antibody.