Anti raf

Celastrol was purchased from Calbiochem (San Diego, CA, USA) and dissolved in DMSO. Cobalt chloride, N-acetylcysteine (NAC) and cycloheximide (CHX) were purchased from Sigma (St Louis, MO, USA). LY294002 was purchased from Alexis (San Diego, CA, USA). The antibodies used were as follows: anti-HIF-1α and anti-HIF-1β (BD Transduction Laboratories, San Jose, CA, USA); anti-Raf, anti-Akt, anti-p-Akt (S473), anti-p-p70S6K (T389) and anti-PARP (Cell Signaling, Beverly, MA, USA); anti-BNIP3, anti-p53 and anti-p21 (Santa Cruz, Dallas, TX, USA); and anti-Bcl2, anti Bcl-xL and anti-β-actin (Calbiochem, San Diego, CA, USA). The dual-Luciferase reporter assay system was purchased from Promega (Madison, WI, USA).

Total protein was extracted from TT, TPC-1, and ARO cells. Western blotting was employed to detect the expression of target proteins. The primary antibodies, including anti-epidermal growth factor receptor (EGFR), anti-phospho (p)-EGFR, anti-H-RAS, anti-RAF, anti-p-RAF (Ser259), anti-MEK1/2, anti-p-MEK1/2 (Ser217/221), anti-extracellular signal-regulated protein kinase 1/2 (ERK1/2), and anti-p-ERK1/2 (Thr202/Tyr204) antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Anti-B-cell lymphoma-2 (Bcl-2), anti-Bcl-2-associated X protein (Bax), anti-cleaved caspase-3 (cas-3), anti-cleaved poly adenosine diphosphate-ribose polymerase (PARP), and anti-β-actin antibodies were purchased from ProteinTech (Chicago, IL, USA). The horseradish peroxidase-conjugated secondary antibodies were purchased from CST. The results were normalized to the expression level of β-actin. The proteins were visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA). The bands were semi-quantified using Image J software.