Horseradish peroxidase secondary

PLX4032 (Vemurafenib) and 4-OHT (4-hydroxy tamoxifen) were purchased from Selleck chemicals and Sigma, respectively. ERK (1:1000), phospho ERK (1:1000), B-Raf (1:1000) primary antibodies and horseradish peroxidase secondary antibodies were from Santa Cruz Biotechnology, Twist1 (1:500) antibody was from R&D systems, and tubulin (1:1000) antibody was from Sigma.

Treated and untreated HosDXR150 cells were lysed in 50 mM Tris/HCl, 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton-X, 0.1 mM Na₃VO₄ plus protease inhibitors (Roche Molecular Biochemicals, Germany). Equal amounts of proteins were resolved on a 7% or 10% SDS-PAGE, transferred onto nitrocellulose membranes, and then successively blocked with 5% non-fat dry milk. Anti-RBL2 was from BD Transduction Laboratories. Anti-FAS, anti-CASP10 and anti-IL12A were from Sigma-Aldrich. Anti-TP73, anti-TP53, anti-actin and the horseradish peroxidase secondary antibodies were from Santa Cruz Biotechnology. Primary antibodies were used at a dilution of 1∶200. Secondary antibodies were used at a dilution of 1∶5000. Signals were acquired using the ImageQuant LAS 4000 (GE Healthcare).