2.9. Radioactive In Vitro Kinase assay

Purified GST-GRAF3-BAR-PH-GAP or GST-GRAF3 BAR-PH-GAP-Y376F were incubated in kinase buffer (25 mM MOPS, 25 mM MgCl₂, 5 mM EGTA, 2 mM EDTA, 12.5 mM β-glycero-phosphate, 2.5 mM DTT) with either active FAK (R&D Systems, Minneapolis, MN, USA) or Src (Sigma, St. Louis, MO, USA) kinase and radioactive γ-³²P (Perkin Elmer, Waltham, MA, USA) at 30°C for 15 min. Reaction mixture was run on a 10% SDS-PAGE gel, gel was dried and phosphorylation was assessed by radiograph. Gel was rehydrated and stained with Coomassie Blue to assess total protein concentration.