Specific horseradish peroxidase conjugated secondary antibodies

Cell were treated with or without ATO for the indicated time and subsequently lysed in the RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Amresco, Cleveland, Ohio) and/or phosphatase inhibitor cocktail (Cell Signaling Technology). The whole cell lysis was subjected to SDS-PAGE, transferred onto a PVDF membrane, and then immunoblotted with respective primary antibodies. The bound primary antibody were visualized with specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) using an enhanced chemiluminescence (ECL) reagents (Thermo Scientific, Hudson, NH). The bar graphs corresponding to the Western blots were generated through densitometric analysis with Image J software.

Proteins extracted from cells or tissues were quantified using Bradford Assay (Bio-Rad). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Bio-Rad). After blocking, the membranes were probed with primary antibodies and then incubated with specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Immunodetection was performed using enhanced chemiluminescence consistent with the manufacturer’s protocol (Thermo Fisher Scientific). Primary antibodies including anti-TAK1, anti-p-TAK1 (T187), anti-JNK, anti-p-JNK, anti-p38, anti-p-p38, anti-Smad3, anti-p-Smad3, anti-p65 and anti-β-actin were purchased from Cell Signaling Technology. Anti-p-TAK1 antibody (T184) was purchased from Thermo Fisher Scientific. Anti-Col I, Anti-Col III and anti-SIRT1 were purchased from Abcam. Anti-p20 antibody was purchased from BioVision (Milpitas, CA, USA). The band intensities were quantified and normalized to the corresponding controls. β-Actin was used as a loading control for internal correction.