Cells were washed and resuspended in RPMI. Cells were incubated at 37°C for 10 to 15 min before treatment with inhibitors (25 or 5 μM 3-IP-PP1) for 1 min. Cells were lysed by adding concentrated lysis buffer to a final concentration of the following: 1% NP-40, NaVO4 (2 mM), NaF (10 mM), EDTA (5 mM), and HALT protease inhibitor cocktail (Invitrogen). Samples were vortexed briefly and placed on ice for 10 min and then centrifuged to pellet debris. Anti-Lck beads [25 μl per immunoprecipitation (IP)] were added and mixed for 1.5 hours at 4°C. Beads were rinsed with ice-cold lysis buffer twice followed by kinase buffer [50 mM Hepes (pH 7.0), 2 mM dithiothreitol, 5 mM MgCl2, 0.2 mM NaVO4, and 0.5 mM β-glycerophosphate]. Beads were resuspended in 60 μl of kinase buffer containing substrate [1 μg of glutathione S-transferase (GST) CD3 ζ, Sino Biological] and adenosine 5′-triphosphate added to 0.2 mM. Samples were incubated with agitation at 25°C for 5 min and placed on ice. Supernatant containing substrate was collected. Phosphorylation status of substrate (GST CD3 ζ) and immunoprecipitated Lck were assessed by immunoblot.

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