Nupage novex midi gel system

Cell lines, tumors from cell-line xenografts, and tumors from patient-derived xenografts (PDX) were prepared and lysed in lysis buffer (20 mmol/L Tris, 150 mmol/L NaCI, 1% NP-40, 1 mmol/L EDTA, 1 mmol/L EGTA, 10% glycerol, and protease and phosphatase inhibitors). The samples were incubated on ice for 30 minutes, then centrifuged at 14,000 rpm for 10 minutes at 4°C. Tumor lysates were homogenized with Tissuemiser (Thermo Fisher Scientific) in the lysis buffer described previously, incubated for 30 minutes on ice, and centrifuged at 14,000 rpm for 10 minutes at 4°C. Protein concentration was determined by BCA Protein Assay (Pierce). Proteins were resolved using the NuPAGE Novex Midi Gel system on 4% to 12% Bis–Tris gels (Invitrogen), transferred to polyvinylidene difluoride membranes (PerkinElmer) in transfer buffer (Bio-Rad) with 20% methanol. Following transferring, the membrane was blocked in PBS-T with 5% nonfat milk for 1 hour and then incubated with the indicated antibodies overnight. After secondary antibody (GE Healthcare) incubation, the antibodies on the membranes were detected with the Syngene G: Box camera (Synoptics). Representative blots are shown in the figures.

Cell lines and tumors from BT-474 xenografts as well as PDXs were prepared and lysed in lysis buffer (20 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 10% glycerol, and protease, and phosphatase inhibitors), incubated on ice for 15 min, and centrifuged at max speed for 10 min at 4 °C. Tumor lysates were homogenized with Tissuemiser (Fisher Scientific) in the lysis buffer described previously, incubated for 20 min on ice, and centrifuged at max speed for 10 min at 4 °C. Equal amounts of the detergent-soluble lysates were resolved using the NuPAGE Novex Midi Gel system on 4–12% Bis–Tris gels (Invitrogen), transferred to polyvinylidene fluoride membranes (PerkinElmer) in between six pieces of Whatman paper (Fisher Scientific) set in transfer buffer from Biorad with 20% methanol, and following transfer and blocking in 5% nonfat milk in PBS, probed overnight with the antibodies listed above. Representative blots from at least three independent experiments are shown in the figures. Chemiluminescence was detected with the Syngene G: Box camera (Synoptics).