An acute, but not germline or conditional, loss of <i>dlg1</i> impairs receptor-mediated actin polymerization.

<p>(A) <i>dlg1^flox/flox</i> or <i>dlg1^flox/flox:CD4^cre</i> CD4⁺ T cells were allowed to conjugate with anti-TCR antibody-coated beads for 20 minutes and stained for actin with rhodamine-phalloidin (<i>top panel</i>). At least 50 cells were scored for actin localization to the T cell/bead interface (<i>bottom panel</i>). (B) Expanded T cells from <i>dlg1^wt;BG</i> or <i>dlg1^ko;BG</i> mice were restimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 5 or 15 minutes. Cells were stained with a combination of anti-CD4, anti-CD8, and FITC-phalloidin and assessed by FACS to determine the relative level of induced actin polymerization in CD4⁺ (<i>top panel</i>) and CD8⁺ (<i>bottom panel</i>) T cell populations. These data are representative of 2 independent experiments, n≥4 for WT and KO samples. (C) Whole cell lysates from Jurkat cells expressing empty vector (empty) or Dlg1 shRNA were immunoblotted with antibodies against Dlg1 or GAPDH, as indicated. (D) Jurkat T cells (green) were transfected with either pCMS3.eGFP.H1p empty vector or pCMS3.eGFP.H1p containing a shDlg1 target sequence. Cells were stimulated with SEE-pulsed (or untreated) Raji B cells (blue) and stained with rhodamine-phalloidin (red) (<i>left panel</i>). At least 50 conjugates were scored for F-actin localization to the T cell/APC interface in each of two independent experiments (<i>right panel</i>). Data represent mean +/− StDev. n.s., not statistically significant.