In vitro kinase assay

Purified His-Pak4cat (55 nM) or His-Pak1cat (55 nM) was mixed with the purified protein of GST, GST-Inka1-iBox, or GST-Inka2-iBox (0.75 ng–400 ng), supplemented with 50 μM DTT and 20 μM ATP. The mixture was then incubated with 1 μg of AKT substrate II peptide (CKRPRAASFAE) as the Pak4 substrate (Promega) or 1 μg of GST-Raf13 as the Pak1 substrate with a total volume of 5 μL in a 384-well plate (PerkinElmer, Waltham, MA, USA) at 30°C for 1 h. For the termination of kinase reaction and depletion of the remaining ATP, 5 μL ADP-Glo reagent (Promega) was added to each reaction mixture and incubated for 40 min at 25°C. Then, a 10 μL kinase detection reagent (Promega) was added and incubated for 1 h at 25°C to convert the generated ADP to ATP. The amount of produced ATP was measured as a luminescence signal of luciferase activity using the Nivo S luminometer (PerkinElmer). The luminescent signal is proportional to the ADP concentration generated and reflects the Pak activity.