Aproptinin

Cells were lysed using either 1.5x Laemilli buffer at a concentration of 10,000 cells/μL or standard RIPA buffer supplemented by 23.4 μM Leupeptin (Roche), 6.1 μM Aproptinin (Roche), 14.5 μM Pepstatin A (Roche), 0.1 mM PMSF (Millipore), and 1 mM Sodium Orthandovate. Samples were then boiled at 100°C for 10 minutes. Extracts were loaded at a range of 50,000–150,000 cells or 5–15 μg of protein and separated on 12% acrylamide gels under denaturing and reducing conditions. Gels were transferred onto 0.45 μm nitrocellulose membranes. Blots were developed using LI-COR's Odyssey imaging system. The following primary antibodies were used: rat anti-HA (Roche) at 1:1000 and rat anti-actin (Abcam) at 1:10,000. Secondary antibodies were as follows: goat anti-rat IRDye 800CW (Li-Cor Biosciences) and goat anti-rabbit IRDye 680RD (Li-Cor Biosciences).

Cell extracts were prepared with lysis buffer at pH 7.4 consisting of 150 mM NaCl, 50 mM Tris, 1% Triton-X100, 23.4 μM Leupeptin (Roche), 6.1 μM Aproptinin (Roche), 14.5 μM Pepstatin A (Roche), and 0.1 mM PMSF (Millipore). Protein concentration was determined with a Micro BCA Protein assay kit (Thermo Scientific). Extracts were separated on 12% acrylamide gels under denaturing and reducing conditions and transferred to nitrocellulose membranes. Blots were developed using standard film methods with HRP conjugated secondary antibodies in conjunction with Luminata Crescendo HRP substrate (Millipore).