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. Author manuscript; available in PMC: 2018 Nov 14.
Published in final edited form as: Circ J. 2018 Jan 19;82(4):1195–1204. doi: 10.1253/circj.CJ-17-0732

Figure 7.

Figure 7.

Phosphorylation of eukaryotic elongation factor-1A1 (eEF1A1) by Rho-associated kinase (ROCK)2. (A) Expression of P-EF1A1, t-eEF1A1 and GAPDH in mouse brain endothelial cells (MBEC) from wild-type (WT), ROCK1+/−, ROCK2+/− and EC-ROCK2−/− (n=3). (B) Increased affinity of phosphorylated eEF1A1 to endothelial nitric oxide synthase (eNOS) mRNA 3’-UTR. GST-eEF1A1 phosphorylated by ROCK2 increased its affinity to eNOS mRNA 3’-UTR (n=5). (C) Phosphorylation of eEF1A1 by ROCK1 and ROCK2 in vitro. Purified ROCK2 protein robustly phosphorylated GST-eEF1A1, whereas purified ROCK1 did so very weakly. The upper band represents phosphorylated GST-eEF1A1 (78 kDa), and the lower band auto-phosphorylated ROCK (ROCK1: 61.4kDa; ROCK2: 63.3kDa). GST, GST only; eEF1A1, GST-tagged eEF1A1; Fas, fasudil (100μmol/L), (n=5). (D) Phosphorylation of eEF1 A1 at Thr432 by ROCK2. Mutagenesis of Thr432Ala (ΔT432A) abolished the phosphorylation of eEF1A1 by ROCK2. The upper band represents phosphorylated GST-eEF1A1 Thr432 and the lower bands the auto-phosphorylated ROCK proteins (caROCK1: 61.4kDa; caROCK2: 63.3kDa). ****P<0.0001. All data are expressed as mean±SEM.