Figure 6. KapB-mediated activation of RhoA-GTPase and PB dispersal requires MK2.

HUVECs were sequentially transduced with two populations of recombinant retroviruses: firstly, puromycin-resistant viruses that express either a short hairpin RNAs (shRNAs) against MK2 or the scrambled (scr) shRNA control, and secondly, viruses that express KapB or the empty vector. After the first step, positive transductants were selected with puromycin for 2 days. A) To examine MK2 expression after knock-down, HUVECs were washed with PBS and lysed in 1x SDS-protein sample buffer containing protease inhibitors and processed for SDS-PAGE and immunoblotting using anti-MK2 and anti-beta-actin. One representative blot of two is shown. B) To assess RhoA activity, transduced HUVECs were assayed for active (GTP-bound) RhoA using the Active Rho Detection Kit (CST) according to manufacturer's instructions. Confluent 6-well plates of HUVECs cells were used for the assay. Total cell lysate (1/10) and pull-downs containing the active (GST-bound) form of RhoA were subjected to SDS-PAGE and immunoblotted with anti-RhoA. One representative experiment is shown. C–D) To examine PBs, transduced cells were seeded onto coverslips. KapB-expressing cells were identified by positive immunoflurescent staining with anti-KapB. To quantify p-body disruption, the number of cells expressing KapB that retained normal p-bodies was counted as for Fig. 4 (n = 3 independent experiments; Scale bar  = 10 µm).