Supplementary Figure 2: Characterization of Lck autophosphorylation and dephosphorylation reactions.
From: In vitro membrane reconstitution of the T-cell receptor proximal signaling network
(a-c) Time course of autophosphorylation of liposome-bound Lck at different ATP concentrations. Panel a, immunoblots and a quantification plot showing the kinetics of Y394 phosphorylation at indicated ATP concentrations. Panel b, immunoblots and a quantification plot showing the kinetics of Y505 phosphorylation at indicated ATP concentrations. Panel c, immunoblots and a quantification plot showing the kinetics of Y505 phosphorylation at indicated ATP concentrations. Unphosphorylated Lck (WT) or Lck (Y394F) was pre-bound to liposomes at ~500 ΞΌm-2 density, and experiments were conducted at RT essentially as described in Online Methods. (d) An immunoblot showing the time-dependent dephosphorylation of Lck Y505 by the cytosolic portion of CD45. Freshly purified His10-Lck (10 ΞΌM) was incubated with 0.5 ΞΌM GST-CD45 on ice for indicated length of time, and then subjected to SDS-PAGE and WB using an mAb against pY505-Lck. (e,f) Immunoblots and quantification plots showing the time-dependent changes in phosphorylation of Lck. 5.04 ΞΌM His10-Lck (WT) was incubated with indicated concentrations of ATP in solution on ice. Aliquots of reactions were terminated with SDS sample buffer and indicated time points and analyzed for phosphorylation on both Y394 (panel e) and Y505 (panel f) using phosphospecifc antibodies (Online Methods).
