Fig. 4. MK2 and MK3 are required for thrombin-induced HSP27 phosphorylation.

(A) Left: Immunoblotting analysis of HSP27 and MK2 phosphorylation in HUVECs pretreated with 300 nM PF3644022 (MK2 inhibitor) or DMSO for 1 hour and stimulated with 10 nM thrombin (α-Th) for the indicated times. Blots are representative of four independent experiments. Right: Densitometric analysis of the fold-change in the relative abundances of the indicated phosphorylated proteins over time. Data are means ± SD of four independent experiments and were analyzed by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Left: In vitro assay of MK2 kinase activity. Immunoblot of HSP27 phosphorylation in HUVECs pretreated with PF3644022 or DMSO and stimulated with 10 nM α-Th. Cell lysates were analyzed by immunoblotting for HSP27 phosphorylation and MK2. Right: Data are means ± SD of four independent experiments and were analyzed by Student’s t test. *P < 0.05. (C) Left: Immunoblotting analysis of HSP27 phosphorylation in HUVECs transfected with MK3-specific or nonspecific (NS) siRNAs, pretreated with PF3644022 or DMSO, and stimulated with α-Th. Right: Densitometric analysis of the fold-change in the relative abundances of the indicated phosphorylated proteins over time. Data are means ± SD of four independent experiments and were analyzed by two-way ANOVA and Dunnett’s post-tests. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Top: Immunoblotting analysis of MK2-MK3 co-immunoprecipitates from HUVECs stimulated with α-Th. Blots of HSP27 and MK2 phosphorylation are representative of three independent experiments. Bottom: Data are means ± SD of three independent experiments and were analyzed by Student’s t-test. **P < 0.01. (E) Phyre homology modeling of HSP27 residues Ser¹⁵, Ser⁷⁸, and Ser⁸² within the predicted 3D structure. The three key serine residues are shown in green, and their positions are illustrated in ribbon (left), space-filled (middle), and merged (right) models.