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. 2016 Sep 23;291(45):23693–23703. doi: 10.1074/jbc.M116.749341

FIGURE 4.

FIGURE 4.

Roles of Plk1 phosphorylation at Thr-210 and vimentin Ser-56 phosphorylation in smooth muscle contraction. A, representative immunoblots (IB) illustrating the expression of WT Plk1 or T210A Plk1 mutant. Extracts of human bronchial tissues transduced with plasmids encoding WT Plk1 or T210A Plk1 mutant were immunoblotted with antibodies against Plk1, GFP, or GAPDH. GFP-Plk1 was detected in the extracts of tissues transduced with plasmid for WT or T210A Plk1. Blots are representative of experiments from three donors. B, contraction of human bronchial rings was evaluated, after which they were transduced with the plasmids as described under “Experimental Procedures.” Contractile responses were compared before and after incubation. *, significantly lower contractile force in bronchial rings expressing T210A Plk1 as compared with tissues transfected with WT Plk1 or untransfected (UT) tissues (p < 0.05). Data are the mean values of experiments from three donors. Error bars indicate S.D. C, cells expressing WT or T210A Plk1 were stimulated with ACh (10−4 m, 5 min) or left unstimulated. Vimentin phosphorylation at Ser-56 in the cells was evaluated using immunoblot analysis. Data are the mean values of experiments from four batches of cell culture. Error bars indicate S.D. (*, p > 0.05). D, myosin light chain (MLC) phosphorylation at Ser-19 in cells expressing WT or T210A Plk1 was assessed by immunoblot analysis. Myosin phosphorylation was similar in cells expressing WT or T210A Plk1 (NS, not significant). Data are the mean values of experiments from four batches of cell culture. Error bars represent S.D. E, representative immunoblots showing the expression of WT and S56A vimentin. Extracts of human bronchial tissues transduced with plasmids encoding WT or S56A vimentin were probed with antibodies against vimentin, GFP, or GAPDH. Blots are representative of experiments from three donors. F, contraction of tissues expressing S56A vimentin is reduced as compared with tissues treated with WT vimentin or UT tissues. Data are the mean values of experiments from three donors. Error bars indicate S.D.