Initial characterization of ezrin transgenic mice. (A) Assessment of ezrin localization at the plasma membrane. Purified peripheral T-lymphocytes were sonicated, the nuclei and cellular debris were removed by centrifugation, and the postnuclear fraction (PN) was separated by ultracentrifugation into the soluble fraction (S100, cytosolic) and the pellet (P100, containing plasma membrane). Samples resolved by SDS-PAGE were analyzed by Western blot with antibodies to HA (to detect transgenic ezrin), to pERM (to detect endogenous phosphorylated ERM; note that this antibody does not detect the T567E ezrin), to ezrin (to detect endogenous and transgenic ezrin), and to Na/K ATPase (to assess plasma membrane enrichment in P100). Note that loading for the P100 fraction was 6 times that of the PN and S100 fractions (18 × 10⁶ vs 3 × 10⁶ cell equivalents) to assure sensitive detection of protein localization there. (B) Lower T-lymphocyte number in lymph nodes in T567E mice (P < .05). Data are mean ± SE of the number of T cells recovered from cervical, axillary, inguinal, lumbar, and mesenteric lymph nodes of 7 individual mice. (C) Reduced in vitro lymphocyte transmigration in T567E mice (P < .05). Primary splenic lymphocytes (20 × 10⁶/mL) were incubated in the upper chamber of transwell chambers with 3-μm polycarbonate membranes, and the number of cells transmigrated to the lower chamber containing 400 ng/mL SDF-1 was assessed after 2 hours by flow cytometry. Data are mean ± SE of transmigration results for 7 T567E mice and 5 WT mice assessed in 4 separate experiments. (D) Slower migration rate of CD4 T-lymphoblasts from T567E mice (P < .0001). Migration was assessed on an ICAM-1–coated surface. Data are mean ± SE of values for 72 WT and 80 T567E cells in 3 separate experiments.