Eps15-KO mice are viable and show an immunological defect. A.

<p>Scheme depicts the region within the Eps15 wildtype (WT) locus that was altered to generate the knockout (KO) locus. The first coding exon of Eps15 and 2 kb of the 5′ promoter region were replaced by gene targeting with a neomycin (NEO) cassette. The location of the primers (a, b, c) used for genotyping is indicated (not to scale). <b>B.</b> Three primer PCR on tail biopsies gave the expected bands for the WT and KO allele: 515 and 642 bp, respectively. <b>C.</b> Western blot of total spleen lysates from Eps15-WT (WT) or Eps15-KO (KO) mice decorated with an anti-Eps15 antibody, or anti-vinculin antibody as loading control, shows lack of Eps15 protein in Eps15-KO mice. <b>D.</b> Bar graph depicting the percent of mice that have been weaned (black bars) for the indicated genotypes (wildtype (WT), heterozygous (Het), knockout (KO)) from the breeding of Eps15 heterozygous mice. The expected frequency for each genotype is depicted by the grey bars. A total of 231 pups were analyzed. <b>E.</b> Western blot analysis of multiple tissues from Eps15-WT (WT) and Eps15-KO (KO) mice reveals ubiquitous expression of Eps15 and Eps15L1. Total tissue lysates (10 µg protein) were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed for Eps15, Eps15L1 or vinculin, as loading control, as indicated. For brain lysates obtained from Eps15-WT (WT) or Eps15-KO (KO) mice, 10 and 20 µg protein were loaded to allow a better comparison of Eps15 and Eps15L1 protein levels. <b>F.–G.</b> Bar graphs displaying cell surface receptor levels (<b>F</b>) and internalization rate constants Ke (<b>G</b>) for the transferrin receptor (TfR) and the EGF receptor (EGFR) in Eps15-WT (white bars) and Eps15-KO (black bars) primary mouse fibroblasts. <b>H.</b> Bar graph depicting the number of white blood cells (WBC), lymphocytes (Lym), monocytes (Mo), granulocytes (Gr) and eosinophils (Eo) in the peripheral blood of 3-month-old Eps15-WT (WT, white bars, n = 10) and Eps15-KO (KO, black bars, n = 10) mice as determined by hematological analysis. <b>I.</b> Bar graph depicting the number of B cells (B), T cells (T), granulocytes (Gr), monocytes (Mo) and natural killer cells (NK) in the peripheral blood of 3 month old Eps15-WT (WT, white bars, n = 10) and Eps15-KO (KO, black bars, n = 10) mice as determined by FACS analysis. Statistical significance was assessed using Student’s t-test. Significant differences are indicated as * = p<0.05, *** = p<0.005.