Dc protein assay reagents package kit

Cells were lysed and sonicated in RIPA lysis buffer (Cell Signaling, Danvers, MA) containing phosphatase inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Lysates were centrifuged at 10,000×g for 10 minutes at 4°C. Pellets were discarded and the total protein concentration of the supernatants was determined by DC Protein Assay Reagents Package kit (Bio-Rad, Hercules, CA). The samples were boiled for 5 minutes at 97°C. 25 µg of protein was loaded per lane for SDS-PAGE gel separation and then transferred to nitrocellulose membrane. Membranes were blocked with Blocking Buffer (Thermo Scientific, Waltham, MA) and then incubated overnight at 4°C with primary antibodies against PTEN, actin (Cell Signaling), or PDCD4 (Rockland Immunochemicals, Limerick, PA). Following the incubation of membranes with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling) for 1 hour at room temperature. Labeled proteins were detected with chemiluminescence detection reagent (Cell Signaling). The membrane was transferred to x-ray film and exposed. The intensity of the protein bands in films was quantified using ImageJ software and the data was reported as % of control.

Following exposure to propofol or DMSO (control), the cells were rinsed with PBS and were lysed and sonicated in RIPA lysis buffer (Cell Signaling, Danvers, MA) containing phosphatase inhibitor cocktail (Roche Diagnostics). Lysates were centrifuged at 10,000×g for 10 min at 4°C. Pellets were discarded and the total protein concentration of the supernatants was determined using a DC Protein Assay Reagents Package kit (Bio-Rad, Hercules, CA). The samples were boiled for 5 min at 97°C. 25 μg of protein was loaded per lane for SDS-PAGE gel separation and then transferred to nitrocellulose membrane. Membranes were blocked with Blocking Buffer (Thermo Fisher Scientific, Waltham, MA) and then incubated overnight at 4°C with primary antibodies against phosphorylated STAT3 (pSTAT3tyr705), STAT3, Sprouty 2, Akt, phosphorylated Akt (pAktser473), and tubulin (Cell Signaling). The membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling) for 1 hour at room temperature and labeled proteins were detected with chemiluminescence detection reagent (Cell Signaling) and obtained on X-ray film. Optical densities were quantified using ImageJ software and the data was reported as % of control.