Figure 2.

Quantification of pY394-Lck and pY505-Lck in T Cells

(A and B) Denatured lysates from (A) Jurkat cells or (B) human CD4⁺ T cells were split into two halves and subjected to three rounds of immunoprecipitations (IP) with pY416 Ab (pY416) or rabbit IgG (Control), respectively. Lysates were analyzed by immunoblotting (IB) for residual pY394-Lck, Lck, and pY505-Lck. The loading control was ZAP-70. IBs were scanned for fluorescence and relevant bands were quantified. Numbers below each panel correspond to the actual fluorescence signals.

(C) Lck isolated from Jurkat cells with the 3A5 mAb was split in two and incubated with (AP) or without (Ctr) alkaline phosphatase for 30 min at 37°C. The fraction of pY394 dephosphorylated was determined by immunoblotting (left panel). Lck from 10⁷ cells (twice for each condition) was in-gel digested with trypsin, after spiking with 1 pmol of [L-C¹³N¹⁵] IEDNEYTAR. Each sample was analyzed twice by MS. Histogram represents mean picomoles ± SE, n = 4 of endogenous LIEDNEYTAR in AP-treated and untreated samples. Details of AQUA and MS are available in the Supplemental Information.