Supplementary Figure 8: Treatment of Jurkat cells with a Lck-specific inhibitor decreased the phosphorylation of Y505.
From: In vitro membrane reconstitution of the T-cell receptor proximal signaling network
(a) Immunoblots showing the effects of Lck specific inhibitor on the phosphorylation of Y505 and Y394 of Lck. Jurkat cells were treated with either 0.2 ΞΌM or 20 ΞΌM cell-permeable Lck specific inhibitor (IC50: 0.016 ΞΌM for Lck and 5.18 ΞΌM for Csk4) or equal volume of DMSO (indicated as β0 ΞΌMβ) at 37 Β°C for 0.5 h. Cells were harvested, lysed and subjected to SDS-PAGE, as described in Online Methods. Phosphorylation of Y505 and Y394 were immunoblotted using mouse anti-pY505-Lck mAb (558552, BD Phosflow) and rabbit anti-pY416-Src polyclonal Ab (2101S, Cell signaling), respectively. Total Lck and GAPDH levels from the same samples were determined by WB using rabbit anti-Lck polyclonal Ab (2984S, Cell Signaling) and mouse anti-GAPDH mAb (MAB374, Millipore). (b) A bar graph showing the quantification of immunoblots shown in a. pY505 and pY394 WB signals were quantified, and normalized to the DMSO control condition. Error bars represent s,d. from triplicate determination. Statistical significance was evaluated by one-sided Student's t test, *** p < 0.001.
