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. 2022 Mar 10;43(24):2317–2334. doi: 10.1093/eurheartj/ehac109

Figure 8.

Figure 8

Rev-erbα interacted with OPHN-1, promoted RhoA activity and phosphorylation of ERM. (A) The protein–protein interaction analysis showed the top-ranked protein. (B) Rabbit anti-Rev-erbα monoclonal antibody was used to immunoprecipitate Rev-erbα from the lysates of resting Rev-erbαfl/flCre− platelets and U46619-stimulated Rev-erbαfl/flCre− platelets. Rabbit IgG was used as the negative control. Mouse anti-OPHN-1 monoclonal antibody was used to immunoprecipitate OPHN-1 from the lysates of resting Rev-erbαfl/flCre− platelets and U46619-stimulated Rev-erbαfl/flCre− platelets. Mouse IgG was used as negative controls. The human platelets were subjected to co-immunoprecipitation assay using the same method (n = 4 independent experiments). (C) Active GTP-bound RhoA in Rev-erbαfl/flCre− and Rev-erbαfl/flPF4Cre+ platelets stimulated with U46619 (0.1 μg/ml) were analysed using G-LISA (n = 5 independent experiments). Data were analysed by two-way ANOVA test followed by Bonferroni post hoc analysis. (D) RhoA protein expression in the Rev-erbαfl/flCre− and Rev-erbαfl/flPF4Cre+ platelets. Quantification of the ratio of RhoA to GAPDH (n = 4 independent experiments). Data were analysed by Student's t-test. (E) Detection of phosphorylated ezrin (Thr567)/radixin (Thr564)/moesin (Thr558)(p-ERM) in Rev-erbαfl/flCre− and Rev-erbαfl/flPF4Cre+ platelets using phospho-specific antibodies. Total amounts of ezrin/radixin/moesin(t-ERM) and GAPDH (loading control) in platelet lysates. Quantification of the ratio of p-ERM to t-ERM (n = 4 independent experiments). Data were analysed by two-way ANOVA test followed by Bonferroni post hoc analysis. (F) SR8278 was dissolved in DMSO. The vehicles were treated with the same concentrations of DMSO, which were 0.1% in platelet suspension. Active GTP-bound RhoA in untreated resting, U46619 (0.5 μg/ml)-treated, and SR8278 (50 μM) + U46619 (0.5 μg/ml)-treated human platelets were analysed using G-LISA (n = 5 independent experiments). Data were analysed by two-way ANOVA test followed by Dunnett's multiple comparisons test. (G) RhoA protein expression in the vehicle controls and SR8278 (50 μM)-treated human platelets. Quantification of the ratio of RhoA to GAPDH (n = 4 independent experiments). Data were analysed by Student's t-test. (H) Detection of phosphorylated ERM in untreated resting, U46619 (0.5 μg/ml)-treated, SR8278 (10 μM) + U46619 (0.5 μg/ml)-treated, SR8278 (30 μM) + U46619 (0.5 μg/ml)-treated, and SR8278 (50 μM) + U46619 (0.5 μg/ml)-treated human platelets using phospho-specific antibodies. Total amounts of ERM and GAPDH (loading control) in platelet lysates. Quantification of the ratio of p-ERM to t-ERM (n = 4 independent experiments). Data were analysed by two-way ANOVA test followed by Dunnett's multiple comparisons test. Veh indicates vehicle.