Horseradish peroxidase conjugated secondary antibody sc 2004

Total cellar protein extraction from different treated MEPM cells using 5 × sodium dodecyl sulfate-lysis buffer supplemented with protease inhibitors (M250; Amresco, Ohio). Protein concentration was determined using a standard BSA protein assay (DingGuo, Beijing, China). Then 40 μg of proteins were fractionated on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk, the membranes were immunoblotted with the primary antibodies: Smad2 (ab71109; Abcam, Massachusetts), p-Smad2 (sc101801; Santa Cruz, California), Smad3 (BM3559; Boster Biotech, Wuhan, China), p-Smad3 (GD-CZ5616 R, Santa Cruz, California), Smad4 (PB0446; Boster Biotech, Wuhan, China), and Smad7 (sc-365846; Santa Cruz, California). -Actin was probed as a loading control. Then membranes were washed and incubated with horseradish peroxidase–conjugated secondary antibody (sc-2004 or sc-2005; Santa Cruz, California). Western blot analysis was performed using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, Nebraska).

Protein expression of the target genes was examined by Western blot. Total protein in the cell lysates was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific) as per the manufacturer's instructions. Equal amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to nitrocellulose membranes and then incubated with anti-Runx2 (M-70, Santa Cruz Biotechnology Inc., Dallas, TX, USA; 1:500 dilution), anti-OC (FL-95, Santa Cruz; 1:500 dilution), anti-BSP (H-157, Santa Cruz; 1:500 dilution), or anti-β-actin (N-21, Santa Cruz; 1:500 dilution) antibody. The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody (Sc-2004, Santa Cruz; 1:40,000 dilution). Immunoreactive proteins were detected using a SuperSignal West Pico Chemiluminescent Substrate blotting kit (Thermo Fisher Scientific). Immunoreactive bands were visualized using chemiluminescence (Syngene GeneGnome, Synoptics Ltd., Cambridge, UK). The relative values of the protein bands were normalized to the internal control β-actin bands (n = 3, for each group).

rhSLPI was purchased from Sino Biological, Inc. (Beijing, China). Medium 200 (M-200-500), Low Serum Growth Supplement kit (S003K-LSGS) and trypsin-EDTA were purchased from Thermo Fisher Scientific, Inc. (Gibco; Waltham, MA, USA). The LDH liquid-UV assay kit was obtained from Human (Wiesbaden, Germany), and MTT was purchased from Ameresco, Inc. (Solon, OH, USA). Antibodies against total p38 (sc-728), phosphorylated-p38 (sc-17852-R), total Akt (sc-8312), phosphorylated-Akt (sc-293125), Bax (sc-6236), Bcl-2 (sc-783), and cleaved caspase 3 (sc-56053), β-actin (sc-130301), and the horseradish peroxidase-conjugated secondary antibody (sc-2004) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Other chemicals were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).