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. 2016 Oct 6;1(16):e88643. doi: 10.1172/jci.insight.88643

Figure 8. Active RhoA is upregulated in type 2B mutant vWF/p.V1316M (2B) megakaryocytes (MKs) and controls the phosphorylation of LIM kinase (LIMK) and cofilin.

Figure 8

(A) Pull-down assays of RhoA-GTP were performed in WT and 2B mature MKs. Western blot and quantification of RhoA and Rhoa-GTP. Statistical significance was determined by Student’s t test. ***P < 0.001, n = 3. (B) Western blot of LIMK and cofilin phosphorylation in WT and 2B MKs in the presence or the absence of the RhoA kinase (ROCK) inhibitor, Y-27632. Mature MKs were incubated for 3 hours with Y-27632 (10 μM) or DMSO (0.2%) and then lysed in SDS denaturing buffer. n = 3. These samples were run in the same gel but the lanes were noncontiguous, as indicated by the separate boxes. (C) Mature MKs after thrombopoietin-induced differentiation in culture were incubated over a fibrinogen matrix for 5 hours in the presence or absence of the ROCK inhibitor (10 μM) or DMSO (0.2%) used as control. Representative images of MKs forming proplatelets. Scale bars: 50 μm. Quantification of the percentage of proplatelet-forming MKs (top graph) and the number of proplatelets/MK (bottom graph) in 3 separate experiments. Statistical significance was determined by 1-way ANOVA followed by Dunnett’s test (50–75 MKs were analyzed/experiment). *P < 0.05, ***P < 0.001.