FIGURE 5.

Csk-dependent co-precipitation of Lck with LYP. A, Lck co-precipitates with Δ288LYP. JTAg cells were transfected with Δ288LYP, and IPs were performed from lysates of resting cells (lanes 1, 3, 5, 7, and 9) or cells stimulated for 2 min with C305 (lanes 2, 4, 6, 8, and 10) using an Ab against Lck (lanes 1 and 2), Fyn (lanes 3 and 4), Itk (lanes 5 and 6), Csk (lanes 7 and 8), or ZAP70 (lanes 9 and 10). B and C, more Lck co-precipitates with LYP-R620 than with LYP-W620. B, JTAg cells were transfected with Δ288LYP-R620 (lanes 1 and 4), Δ288LYP-W620 (lanes 2 and 5), or empty vector (lanes 3 and 6), and IPs were performed from lysates of resting cells using an Ab against Lck (lanes 1–3) or Csk (lanes 4–6). C, JTAg cells were transfected with LYP-R620 (lane 1), LYP-W620 (lane 2), or empty vector (lane 3), and IPs were performed from lysates of resting cells using an anti-Lck Ab. D–F, the co-precipitation of Lck with LYP is dependent upon Csk activity. D, JTAg cells were co-transfected with LYP-R620 (lanes 1–3 and 7–9) or LYP-W620 (lanes 4–6 and 10–12) and with RNA interference oligonucleotides specific for Csk (lanes 2, 5, 8, and 11) or nontargeting ones (lanes 1, 3, 4, 6, 7, 9, 10, and 12). The cells were left unstimulated (lanes 1–6) or stimulated for 2 min with C305 (lanes 7–12). The cell lysates were subjected to IP using an anti-Lck Ab. E, thymocytes were isolated from Csk conditional KO mice (lanes 2 and 4) and control littermates (lanes 1 and 3). The cells were left unstimulated (lanes 1 and 2) or stimulated with anti-CD3 + anti-CD4 for 1 min (lanes 3 and 4). The cell lysates were subjected to IP using an anti-PEP Ab. F, JTAg cells stably overexpressing Δ288LYP-R620 were transfected with HA-Csk (lane 1) or decreasing amounts (3, 2, or 1 μg of plasmid DNA) of the catalytically inactive mutant HA-Csk K222R (lanes 2–4) or vector alone (lane 5). The cells were stimulated for 2 min with C305, and IPs were performed from lysates using an anti-Lck Ab.