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. 2014 Jul;28(7):3249–3260. doi: 10.1096/fj.13-245142

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Figure 5. — Role of p44/42 MAPK mechanism in CS-induced GEF-H1 activation. A, B) Pulmonary ECs were pretreated with U0126 (5 μM, 30 min) before CS stimulation. A) GEF-H1 activation was evaluated by GEF pulldown assay. Western blot detection of GEF-H1 in corresponding total lysates was used as normalization control. B) GEF-H1 was immunoprecipitated under denaturing conditions, followed by probing with phosphothreonine-specific antibody and reprobing with GEF-H1 antibody. C, D) Cells were transfected with GEF-H1 wild-type (GEF-H1-FL) or phosphorylation-deficient mutant (GEF-H1T678A), followed by 18% CS, 15 min. C) Rho activation was evaluated by RhoGTP pulldown assay, D) MLC and p42/44 MAPK phosphorylation was analyzed by Western blotting with corresponding antibody. GEF-H1 transfection efficiency was verified by probing with GFP tag antibody. Bar graphs depict the quantitative analysis of Western blot densitometry data. *P < 0.05; n = 5.