TAB4 triggers cell death of activated mouse T cells and recognizes mouse PSGL-1. (A) Activated mouse T cells on day 5 from stimulation with ConA (2 μg/mL) were incubated with TAB4 or control antibodies plus R anti-H overnight. Following double staining with Annexin-V FITC and propidium iodide, cells were analyzed by flow cytometry to determine the percentage of dead cells. Without gating, 10% of 10 000 total events were displayed. (B) TUNEL assays of control cells (left) and TAB4-treated cells (right). Few TUNEL-positive cells could be found in the activated T-cell (AT) population with IL-2 treatment (left). Plenty of TUNEL-positive cells were observed in AT treated with TAB4 for 6 hours. Arrows: cell nuclei with TUNEL-positive staining. Scale bar: 20 μm. (C) PSGL-1 transfectant cells, 10A, were stained with 5 μg/mL TAB4 or control antibody, hamster Ig, and analyzed by flow cytometry. (D) Total cell extract of 5 × 10⁷ activated mouse T cells at late activated stages were immunoprecipitated by anti–PSGL-1 antibodies, TAB4, and 2PH1, respectively, and followed by immunoblotting for TAB4. (E) Total cell extracts of 3 × 10⁷ activated mouse T cells at late activated stages were depleted for PSGL-1 by 2 successive rounds of incubation with affinity-purified anti–PSGL-1 antibodies using TAB4 or 2PH1. Residual proteins of the depleted extracts were immunoblotted with 2PH1 under reducing conditions. The Zeiss Axioskop was equipped with a 40×/0.75 Zeiss Plan-Neofluar objective lens (Carl Zeiss, Oberkochen, Germany) and a Nikon D1× digital camera (Nikon, Tokyo, Japan). Images were acquired with Nikon Capture 2 software and processed in Adobe Photoshop 7.0 (Adobe, San Jose, CA).