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. 2010 Jun 25;32(6):766–777. doi: 10.1016/j.immuni.2010.05.011

Figure 4.

Figure 4

Catalytic Activity of Dpho-Lck

(A) Lck activity is mostly associated with pY394-Lck. Jurkat lysates were immunoprecipitated with anti-pY416 or anti-Y416 or with control IgG (Control) and subjected to in vitro kinase assay on rCD3-ζ-GST. The reaction mixture was immunoblotted for pY142-ζ, GST, pY394 (pY416), and Lck and quantified by near-infrared fluorescence (numbers below each panel). Overlapping Lck and pY416 fluorescence (not shown) provide positive identification of pY394-Lck. Kinase activity (right panel) was expressed as pY142-ζ signal subtracted for IgG control and normalized for rCD3-ζ-GST.

(B and C) Dpho- and pY394/Y505-Lck have similar kinase activity. As shown in (B), DPho (pY394-pY505) and pY394 (pY394-Y505) were individually enriched by immunoprecipitation (see Results). An aliquot was subjected to dual fluorescence immunoblotting with anti-pY416 (red) and anti-Y416 (green) or anti-pY505 (red) and anti-Lck (green). The control was rabbit IgG. In (C), Dpho- and pY394-Lck from (B) and IgG control immunoprecipitations were assayed for kinase activity on rCD3-ζ-GST as in (A) and immunoblotted for pY142-ζ, GST and pY394 (pY416). Identification of pY394-Lck was as in (A). A representative experiment of three giving similar results is shown.