IVK assay.

An in vitro kinase (IVK) assay was performed to activate p38α by TAB1(384–412), p38α by MKK6, and ATF2 and TAB1 by p38α. p38α (3 μM) was activated with 15 μM TAB1, 3 μM p38α was activated with 0.6 μM MKK6dd, and 0.86 μM ATF2 or TAB1 was activated with 0.2 μM p38α. The reaction was carried out in 1× kinase buffer (25 mM Tris-HCl [pH 7.5], 5 mM β-glycerolphosphate, 2 mM dithiothreitol [DTT], 0.1 mM Na₃VO₄, and 1 mM MgCl₂). ATP (550 μM) was added to the incubation mixture to start the reaction at 37°C for 2 h. At the end of the reaction, samples were collected with 2× sample buffer (20% glycerol, 6% SDS in 0.12 M Tris [pH 6.8], 10% 2-mercaptoethanol, and 0.4% bromophenol blue), heated to 95°C for 10 min, and run on 10% SDS gel under denaturing conditions. The samples were transferred onto a polyvinylidene difluoride (PVDF) membrane using semidry technique, blocked with 4% milk plus 1% bovine serum albumin (BSA) for 1 h at room temperature, and probed with an appropriate primary antibody overnight at 4°C. The blots were probed with an appropriate secondary antibody linked with horseradish peroxidase (HRP) and visualized by enhanced chemiluminescence. The antibodies used included anti-dually phosphorylated p38 (Thr180/Tyr182) (M8177; [Sigma] and 9211; Cell Signaling Technology [CST]) at 1:5,000, total p38 (9212; CST) at 1:10,000, anti-phosphorylated TAB1 (Phil Cohen group, Dundee) at 1:2,000, total TAB1 (Phil Cohen group, Dundee) at 1:4,000, anti-phosphorylated ATF2 (9221; CST) at 1:4,000, total ATF2 (20F1; CST) at 1:8,000, and anti-phosphorylated MKK3/6 (9231; CST) at 1:1,000.