Pf431396

Jurkat T cells were starved in RPMI serum free medium with 0.5% FBS
for 2 h and incubated with DMSO or 400 nM of FAK inhibitor PF431396 or Src
inhibitor RK24466 (Cayman Chemical, Ann Arbor, MI USA) at 37°C for 1
h, and then stimulated by 2.5 μg/ml anti-CD3 antibodies (Abcam,
Cambridge, MA USA) for 10 min. The cells were lysed in 200 ul of lysis
buffer (1% Triton X-100, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2,
0.25 mM ATP) supplemented with protease/ phosphatase inhibitor cocktails.
Clarified lysate was incubated with 10 μg of GST-RA-PH for 1 h at 4
°C with gentle shaking. GST beads were then added to mixture of
lysate and GST-RA-PH and gently agitated for 1 h at 4 °C. Samples
were eluted in Laemmli sample buffer and boiled to 95°C for 10 min.
For In vitro kinase assays, purified RIAM-NT or RA-PH were
incubated with 100 nM FYN (Carna Biosciences) or 50 nM LCK (Carna
Biosciences) in the reaction buffer (5 mM ATP, 20 mM MgCl2, 20 mM Tris-HCl
(pH 7.5), 100 mM NaCl, 50 μM Na₃VO₄ at room
temperature. Reactions were stopped by adding EDTA to the samples to a final
concentration of 50 mM. Samples were separated by 10-15% SDS-PAGE gels.