Figure 2: JKAP directly interacts with and dephosphorylates Lck.

(a) Immunoprecipitation with anti-JKAP and then immunoblotting with anti-Lck of lysates from murine splenic T cells. NS, normal serum. (b) Confocal microscope analysis of JKAP and Lck in stimulated with anti-CD3 (5 μg ml⁻¹, 5 min) and then subjected to double immunostaining with Atto565-congugated anti-JKAP antibody (red) and anti-Lck antibody (green). r, Pearson’s correlation coefficients (mean±s.e.m.) are shown. Scale bar, 10 μm. (c) In vitro binding assays of purified Flag-Lck and GST-JKAP or GST proteins. Left, anti-Flag immunoprecipitation; right, GST-pulldown. (d) FRET analysis of the direct interaction between CFP-JKAP and YFP-Lck in live Jurkat T cells. (e) In vitro phosphatase reactions of JKAP using purified Flag-Lck WT, Y394F or Y505F proteins as the substrates. CS, the catalytically inactive JKAP-C88S mutant. (f) Immunoblotting of p-FAK (Y397) and p-FAK (Y861) in murine splenic T cells on anti-CD3 (5 μg ml⁻¹) stimulation. Relative fold changes were normalized to total FAK protein levels and are shown at the bottom of each panel. (g) [³H]thymidine incorporation assays in GFP⁺-T cells that were sorted from murine splenic T cells transfected with empty vector (pCMV6-GFP) or ectopic GFP-JKAP. Data are presented as means±s.d. Two-tailed Student’s t-test, *P<0.05. Data are representative of four independent experiments. WT, wild-type; KO, JKAP-knockout.