Samples were processed according to the manufacturer’s instructions. WT and Pkd2cKO cell lysates were immunoprecipitated overnight by gentle rotation at 4°C with an anti–B-Raf or an anti–Raf-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) covalently coupled to protein A/G Plus agarose beads. Immunoprecipitates were resuspended in 20 μL of a solution containing 0.5 mM β-glycerophopshate (pH 7.3), 1.5 mM ethylene glycol tetraacetic acid, 1 mM dithiothreitol, and 0.3% Brij 35. The kinase activities of B-Raf and Raf-1 were assessed by the phosphorylation of exogenous

mouse MEK, a natural substrate for the kinases.20 The kinase assay was performed selleckchem in 20 μL of a solution containing 16 μL of 50 mM MgCl2, 2 μL of 1 mM ATP, and 2 μg mouse MEK-1 fusion protein (SignalChem, Richmond, British Columbia, Canada), mixed with 20 μL of the resuspended beads and incubated for 30 minutes. The reaction was stopped by adding sodium dodecyl sulfate sample buffer. Trametinib nmr The reaction product was immunoblotted using an antibody against phosphorylated

MEK (Santa Cruz Biotechnology) and visualized using an enhanced chemiluminescence system. Results are presented as the mean ± SD. Statistical comparisons were made using a Student t test or one-way ANOVA, where more than two groups were compared. Statistical analysis was performed using SAS software (SAS, Cary, NC), and P < 0.05 was considered significant. Pkd2cKO mice were Amoxicillin treated for 8 weeks with 20 or 60 mg/kg/day of sorafenib tosylate, beginning 1 week after the deletion of PC2 gene with tamoxifen. Pkd2cKO mice receiving vehicle with the same schedule after the induction, served as controls. When given at 20 mg/kg/day, sorafenib was relatively well tolerated (8 out of 10 mice survived and showed no clinical

sign of toxicity except for a mild reduction in total body weight (Supporting Fig 1). On the contrary, when administered at 60 mg/kg/day, mice showed significant toxicity, with only 5 out of 10 mice surviving the 8 weeks treatment. The area of the liver cysts was measured as described7, 8 using pancytokeratin and K19 as epithelial markers. Unexpectedly, mice treated with sorafenib showed a significant increase in cystic area compared with control Pkd2cKO mice (Fig. 1B) (Pkd2cKO vehicles: 30,718 ± 5,818μm2 [n = 9] versus 43,228 ± 7,508 μm2 in Pkd2cKO mice treated with 20 mg/kg/day [n = 8], P < 0.001, and 38,695 ± 6,659 μm2 in mice treated with 60 mg/kg/daily [n = 5]). Similarly, the percentage amount of the total area of the lobe covered by K19-positive structures was higher in sorafenib-treated mice than in control mice (Pkd2cKO vehicles: 4.1 ± 0.