Secondary antibody conjugated to horseradish peroxidase

CCRCC cells were lysed in buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.1% (w/v) SDS, 0.1% (w/v) SDC, 1% (v/v) Nonidet P-40, 5 mM NaF, 1 mM Na₃VO₄, 2.5 mM Na₂HPO₄, and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and tissues were lysed in Pro-prep buffer (Intron, Daejeon, Korea). Equal amounts of proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were probed with antibodies to PTEN (Cell Signaling, 91885) and GAPDH (sc-20357, Santa Cruz Biotechnology, Inc.), followed by incubation with the appropriate secondary antibody conjugated to horseradish peroxidase (ThermoFisher Scientific, Waltham, MA). Immunoreactivity was visualized using Immunobilon Western Chemiluminescent HRP Substrate (Millipore) and the Amersham Imager 600.

The AFB₁ used in the study was purchased from Sigma-Aldrich, USA (cat no A6636) and dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 3200 μM. The AFB₁ stock solution was divided into aliquots, wrapped in aluminium foil and stored frozen at −20 °C until used. The AFB₁ stock solution was diluted to the desired concentration in normal growth medium when necessary. The foetal bovine serum (FBS) was purchased from Sigma-Aldrich, USA. Dulbecco’s Modified Eagles Medium (DMEM) (high glucose, L-glutamine, sodium pyruvate and 25 mM HEPES) was purchased from Science Cell. Minimum essential medium (MEM) non-essential amino acids was purchased from Sigma Aldrich, USA while penicillin-streptomycin was purchased from Gibco by Invitrogen, UK. Lipofectamine 2000 was purchased from Gibco by Life Technologies, UK (cat no 11668-019). The human recombinant interferon-alpha 2 (rIFN-α2) was purchased from PBL interferon source (cat no 11115-1). The stock solution of the human recombinant interferon-alpha 2 was diluted to working concentration using phosphate buffered saline containing 0.1% bovine serum albumen as a diluent. The STAT1 (cat no PA5-34504) and GAPDH (cat no QE 212271) primary antibodies were purchased from Thermo Scientific, USA. The secondary antibody conjugated to horse-radish peroxidase (cat no 31430) was purchased from Thermo Scientific, USA.