Pkcζ h 1

For F-actin polymerization, T cells from Itk^-/-, PI3Kγ^-/-, and WT mice were starved for 1.5 h in a serum-free medium. Where noted, cells were pretreated with 0.5 µM Wortmannin (Calbiochem) for 1 h at 37°C. CCL21 (100 nM; Peprotech) was added to 2 x 10⁶ cells/ml in a 37°C water bath, and cells were fixed at indicated time points in 4% cold PFA. Cells were permeabilized and labeled with FITC-Phalloidin (Invitrogen) as described (19 (link)). For polarization assays, T cells were added on 1 µg/ml fibronectin-coated glass slides before the addition of CCL21 (100 nM) and fixation after 20 min with 2% PFA. After permeabilization with 0.1% Triton X-100 (5 min), cells were incubated with anti-protein kinase ζ; (PKCζ) (H-1; Santa Cruz Biotechnology) and biotin-labeled anti-CD44 (IM7; BD Pharmingen) mAbs. Primary antibodies were detected with a Cy3-labeled anti-mouse Ab (Jackson ImmunoResearch Laboratories) and Alexa488-labeled avidin (Molecular Probes). For chemotaxis assays, T_N was pretreated with inhibitors for DOCK2 (CPYPP) and PI3Kγ (AS-605240; both Tocris) as indicated for 1 h at 37°C, and 0.5 x 10⁶ T_N/well were added to Transwell chambers (5-µm pore size; Costar) to migrate to 50 nM CCL21 for 1.5 h at 37°C in the presence of the inhibitors. Cell viability was assessed by PI staining at the end of the experiment. Input and migrated T cells were enumerated by flow cytometry.