4.5. Western Blot Analysis

Protein expression was measured using western blotting as previously described with some modifications [21,46]. Cells were harvested, washed after the indicated treatments, and then lysed. The total protein from these cells was extracted and then separated on SDS-polyacrylamide gels using electrophoresis and detected using specific antibodies. β-Actin was used as an internal loading control. The immobilized proteins were then transferred onto polyvinylidene difluoride membranes and probed using antibodies. Immunoreactive proteins were detected using the ECL western blotting detection system (Fujifilm, LAS-4000, Tokyo, Japan), and signal strengths were quantified using a densitometric program (MultiGauge V2.2).