Secondary antibodies coupled to horseradish peroxidase

Cells were seeded in 6-well plates at a density of 0.5 × 10⁶ per well for 24 hours, followed by incubation with vehicle control, ridaforolimus (15 nM), vorinostat (500 nM), or their combination for 72 hours. Cells were rinsed with PBS, then scraped in lysis buffer (Cell Signaling Technology; Danvers MA), supplemented with protease and phosphatase inhibitor cocktails (Roche Applied Science; Indianapolis, IN). Lysates were centrifuged for 5 minutes at 13,000 × g and quantified using the Protein Assay Reagent from Bio-Rad (Hercules, CA). Equal amounts of proteins (35 μg) were loaded onto 10% NuPage gels (Invitrogen; Carlsbad, CA). Following transfer, blocking, and incubation with primary and secondary antibodies, proteins were visualized by electrochemiluminescence (Perkin-Elmer; Boston, MA) and exposed to HyBlot CL films (Denville Scientific; Metuchen, NJ). Phospho-serine 473 AKT and total AKT antibodies were obtained from Cell Signaling Technology (Danver, MA). β-actin was purchased from Sigma-Aldrich (St. Louis, MO). Secondary antibodies coupled to horseradish peroxidase were obtained from GE HealthCare (Piscataway, NJ). Densitometric semi-quantification of bands was conducted using the ImageJ software (National Institutes of Health). Levels of p-AKT-ser473 bands were normalized to their corresponding total-AKT bands, which were normalized to their corresponding β-actin bands.