Figure 6.
Csk is essential for the suppression of TCR-induced ZAP-70 activation by LAIR-1. A, Jurkat cells, a control CRISPR (clone CO-2), or Csk CRISPR (clones PO-61 and PO-101) cells were examined for Csk protein expression by Western blotting analysis (left panel) or for Csk mRNA by RT-PCR (right panel). Actin was used as control. The data presented are representative of three separate analyses. B, Jurkat cells, a control CRISPR (clone CO-2), or Csk CRISPR (clones PO-61 and PO-101) cells were pretreated with medium (no) or type II collagen (α1(II)) overnight and then stimulated with anti-CD3 and anti-CD28 antibodies for the indicated time period. Whole-cell lysates were collected and analyzed for activation of ZAP-70 by Western blotting analysis using anti–phospho-ZAP70 Tyr-493 (α-pZAP70) antibody. The membrane was stripped and reblotted with non–phospho-specific antibody for ZAP-70 (α-ZAP70). These data are representative of three separate analyses. C, CD4+ T cells from CskAS mice were pretreated with either vehicle (no), PP1* (3-IB-PP1, 10 μm), αI(I) (200 μg/ml), or PP1* + αI(I) overnight and stimulated with anti-CD3 antibody (10 μg/ml) for 5 min. Whole-cell lysates were prepared and analyzed for activation of ZAP-70 by Western blotting analysis using an anti–pZAP-70 Tyr-493 antibody. The membrane was stripped and reblotted with a non–phospho-specific antibody ZAP-70 (α-ZAP70). In the absence of α-CD3, none of these agents were able to elicit T-cell activation (data not shown). The data shown are representative of three separate analyses.