Hrp labeled goat anti rabbit igg

Kidney tissues were lysed and homogenized using the radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl, pH 8.0, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 M NaF plus protease inhibitor cocktail [Sigma-Aldrich] and phosphatase inhibitor cocktail [Sigma-Aldrich]). Extracted protein samples were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (GVS S.p.A.). After blocking with 5% bovine serum albumin or skim milk for 30 minutes, the membranes were incubated with antibodies against α-smooth muscle actin (α-SMA, 1:20,000; Sigma-Aldrich), phosphorylated SMAD3 (p-SMAD3, 1:2,000; Abcam), p-SMAD2 (1:2,000; Abcam), t-SMAD 2/3 (1:2,000; Abcam), 4-hydroxynonenal (4-HNE, 1:2,000; Abcam), Ly6G (1:2,000; Fisher Scientific), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:10,000; Bioworld Technology) overnight at 4 °C. The membranes were then treated for 1 hour at room temperature with horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin G (IgG, 1:3,000; Bethyl-Laboratories) or HRP-labeled goat anti-rabbit IgG (1:3,000; Bethyl-Laboratories). Total protein expression levels were normalized to GAPDH. Band intensities were analyzed using ImageJ software.