Alteration of Methotrexate Biliary and Renal Elimination during Extrahepatic and Intrahepatic Cholestasis in Rats

Activation of TGF-β Receptors and Smad Proteins by Atorvastatin is Related to Reduced Atherogenesis in ApoE/LDLR Double Knockout Mice

“…Mouse aorta samples were homogenized in RIPA lysis buffer (Sigma Chemical Co.), as described previously 31) . Homogenates (15 g aorta proteins) were used for membrane preparation, as described previously 31) .…”

“…Homogenates (15 g aorta proteins) were used for membrane preparation, as described previously 31) . The membranes were blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween-20 (T-BST), and then incubated with primary antibodies at the following concentrations: Goat polyclonal antibody anti-Endoglin (95 kDa) at 1:500 and rabbit polyclonal antibody anti-eNOS (140 kDa) at 1:200 were purchased from Santa Cruz Biotechnology, Inc., rabbit polyclonal antibody anti-ACVRL1/ALK-1 (56 kDa) at 1:500, rabbit polyclonal anti-Smad-2 (56 kDa) at 1:100, rabbit polyclonal anti-pSmad-1 (52 kDa) at 1:200 and rabbit polyclonal anti-VEGF (43 kDa) at 1:1000 were from Abcam plc, rabbit polyclonal anti-TGF R (ALK-5) (53 kDa) at 1:500 was from Sigma Chemical Co., and rabbit monoclonal anti-Smad-1 (58 kDa) at 1:500 and rabbit polyclonal anti-pSmad-2 (52 kDa) at 1:500 were from Millipore.…”

“…As the loading control for Western blot, rabbit polyclonal anti--actin antibody (42 kDa) at 1:5000 was purchased from Sigma Chemical Co., horseradish peroxidase conjugated rabbit anti-goat IgG at 1:5000 was purchased from Pierce Biotechnology (Rockford, USA) and horseradish peroxidaselinked F(ab')2 fragment donkey anti-rabbit IgG at 1:2000 was from GE Healthcare (Prague, Czech Republic). The chemiluminescent process and quantification of immunoreactive bands on the exposed films were carried out as described previously 31) . Equal loading of proteins onto the gel was confirmed by immunodetection of beta-actin.…”

Cholesterol Effects on Endoglin and Its Downstream Pathways in ApoE/LDLR Double Knockout Mice

“…38 Homog-Endoglin, Cholesterol and Atherogenesis enates (15 μg of aorta proteins) were used for membrane preparation as described previously. 38 The membranes were blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBST), and then incubated with primary antibodies at the following concentrations: goat polyclonal antibody anti-endoglin directed to the endoglin (90-95 kDa) at 1:500, rabbit polyclonal anti-TGFβ-RI (ALK-5) directed to the TGFβ-RI (53 kDa) at 1:500, and anti-eNOS directed to the eNOS (140 kDa) at 1:200 (Santa Cruz Biotechnology, CA, USA), rabbit polyclonal antibody anti-ACVRL1/ ALK-1 directed to the ACVRL1/ALK-1 (56 kDa) at 1:500, anti-p-Smad1 directed to the p-Smad1 (52 kDa) at 1:200, and anti-VEGF at 1:1,000 (Abcam, UK), rabbit polyclonal antip-Smad2 directed to the p-Smad2 (58 kDa) at 1:500 (Millipore, USA). As the loading control for western blotting, we used rabbit polyclonal anti-β-actin antibody (42-45 kDa) at 1:5,000 (Sigma Chemical Co), horseradish peroxidase conjugated rabbit anti-goat immunoglobulin G at 1:5,000 (Pierce Biotechnology, Rockford, IL, USA) and horseradish peroxidase-linked F(ab')2 Fragment donkey anti-rabbit antibody at 1:2,000 (GE Healthcare, Prague, Czech Republic).…”

The Effect of Resveratrol on Bone Status in Rats with Bile Duct Obstruction

“…Chronic cholestasis was induced by surgical bile duct obstruction (BDO) according to Brcakova et al [20]. The cannula was inserted into the bile duct, closed and fixed subcutaneously.…”