In vitro kinase assay

TAP-purified MAP4K4 eluted in standard lysis buffer with protease and phosphatase inhibitors were added to kinase assay buffer (25 mM Tris-HCl pH 7.5, 5 mM β-glycerophosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate and 10 mM MgCl₂) containing 20 μM ATPγS (Abcam) and 1 μg of myelin basic protein (MBP) (Sigma). Where specified, ATPγS was left out of the reaction as a negative control. Kinase reactions were carried out as previously described (Allen et al., 2007). Reactions were carried out at 30°C for 30 min. P-nitrobenzyl mesylate (PNBM) (Abcam) was then added (2.5 mM final) and the reaction was incubated at room temperature for 2 hr, followed by addition of 6x SDS loading buffer, boiling of samples, SDS-PAGE and subsequent immunoblotting for phosphorylated MBP. Relative activity was calculated as the ratio of the band intensities (measured with ImageJ) between the thiophosphate ester signal (phospho-MBP) and HA signal (NTAP-MAP4K4).