Fig. 1

Experimental verification of mutual antagonism between NO and RhoA in epithelial (MDCK) cells. (A and B) NO mediates HGF-induced RhoA-Ser188 phosphorylation. MDCK cells were induced with HGF 1 ng/mL for the specified durations with or without a pre-treatment for 20 min with 50 μM L-NAME, an inhibitor of constitutive NOS. A Western blot analysis with pSer188-RhoA and B Quantification of band intensity. The phospho-RhoA-Ser188 bands in the western blot were quantified using ImageJ and normalized with respect to total RhoA intensity. C, D, E and F The Rho effector, ROCK inhibits p-Akt/p-eNOS signaling. MDCK cells were induced with HGF 1 ng/mL for the specified durations with or without 20 min pre-treatment with 20 uM of ROCK inhibitor Y-27632. C Western blot analysis for shorter durations of HGF treatment. Additional timepoints appear in Additional file 1. Commercial antibodies do not detect the canine isoform of eNOS but phospho-eNOS antibodies are reactive to the canine isoform. Quantification of the bands in the western blot: D phospho-Akt-Ser473 normalized against total Akt, E phospho-Akt-Thr308 normalized against total Akt, and F phospho-eNOS normalized against GAPDH. The white line between lanes denotes that a single gel was cropped to remove other treatment conditions in the middle lanes. (Full gel appears in Additional file 1). **p < 0.005