Actin clone 1 19

50 × 10⁶ cells were lysed for 30 min at 4°C in lysis buffer (100 mM Tris-HCl pH 8, 10 mM EDTA, 10 mM NaCl) supplemented with protease and phosphatase inhibitor cocktail tablets (Roche) and samples were centrifuged at 13,000 rpm for 30 min at 4°C. Protein concentrations of supernatants were determined using Bio-Rad protein assay (BIO-RAD). Equivalent protein extract (∼80μg) for each sample was separated by SDS-PAGE and transferred to nitrocellulose membrane using Iblot Gel Transfer stacks and Iblot system (Invitrogen). Membranes were blocked in TTBS (137 mM NaCl, 2 mM KCl, 25 mM Tris and 0.1% tween 20) supplemented with 5% non-fat milk and incubated with primary antibodies against Cleaved Notch1 (Val1744) Antibody (Cell signaling Technology) or Actin (clone I-19, Santa Cruz Biotechnology Inc.) overnight at 4°C with agitation. The fluorescent secondary antibodies, Anti-rabbit or -goat conjugated to CF770 (Biotium) were added for 1 hr at room temperature. Immunoblots were revealed using an Odyssey infrared imaging system (Li-Cor Biosciences) and stripped using Restore Western blot stripping buffer (Pierce).

BSC40 cells (Cat# CRL-2761) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Murine embryonic fibroblasts (MEFs) were a kind gift from Dr. Carl Atkinson. LLC-A9F1 (A9F1) cells were a kind gift from Dr. Mark Rubenstein. All cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum and 1x Penicillin-Streptomycin-L-Glutamine (Corning, Oneonta, NY, USA). Cell viability was measured using the CellTiter-96 Non-Radioactive Cell Proliferation (MTT) assay (Promega, Madison, WI, USA) according to the manufacturer’s recommendations. Antibodies used for this study were purchased from Santa Cruz Biotech (Santa Cruz, CA) and include: actin (clone I19) and c-Myc (clone 9E10).