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. 2015 Jan 8;11(1):e1004597. doi: 10.1371/journal.ppat.1004597

Figure 2. KapB requires the kinase MK2 and RhoA-GTPase to induce actin stress fibers in primary endothelial cells.

Figure 2

A) HUVECs expressing KapB (panels a–d) or controls (MK2-EE [panels e–h] or hsp27-DDD [panels i–l]) were treated with inhibitors to examine the roles of the kinases p38 and MK2 and the GTPase RhoA in stress fiber formation. Cells were either treated for one hour with 3 µM of the p38 kinase inhibitor SB203580, 10 µM of the MK2 inhibitor, Inhibitor III, 10 µM of the rho-associated kinase (ROCK1/2) inhibitor, Y-27632, or DMSO as a vehicle control, before fixation and staining as above. B) To examine activation of the RhoA GTPase by KapB and controls, HeLa-Tet Off cells were transfected with an expression plasmids for KapB, MK2-EE, hsp27-DDD or an empty vector control and assayed for active (GTP-bound) RhoA using the Active Rho Detection Kit (CST) according to manufacturer's instructions. Before lysis, transfected cells were starved in low serum media for a total of 24–28 hours and either treated or not treated with the RhoA activator lysophosphatidic acid (LPA) for 3 minutes. Total cell lysate and pull-downs containing the active (GST-bound) form of RhoA were subjected to SDS-PAGE and immunoblotted with anti-RhoA (CST). One representative experiment of three is shown.