Conjugated to horseradish peroxidase

To assess the efficacy of TRaM and free rapamycin on inhibiting MCEC mTOR activity, MCECs were again exposed to simulated cold hypoxic reperfusion injury, using the model outlined above. In these experiments, UW solution was augmented with TRaM, empty micelles, free rapamycin, or DMSO vehicle. Following 6, 12, 18, 24, or 72 hours of reperfusion, MCEC protein lysates were collected and analyzed by western blot as previously described.^{14 (link)} Briefly, cells were washed, detached mechanically, pelleted and lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, MA) that contained 1× phosphatase and 1× protease cocktail inhibitors (Thermo Fisher Scientific, MA). After quantification, proteins were denatured and separated using 4–20% gradient SDS-PAGE (Bio-Rad Laboratories, CA), followed by wet transfer to a PVDF membrane (Bio-Rad Laboratories, CA) for immunoblotting. Primary antibodies against P-p70S6K (Thr389), p70S6K (all from Cell Signaling, MA), beta actin (Santa Cruz, TX) were used at a 1 : 1000 dilution. Secondary antibodies conjugated to horseradish peroxidase (GE Healthcare, Little Chalfont, UK) were used at a 1 : 2000 dilution. Signal was determined by chemiluminescence using film development and digital imaging methodologies. Quantification of band intensity was performed using ImageJ v1.46 (NIH, MD).