Complete mini protease inhibitor tablet

Rat FL-Jag1-HA (pBOS-SN3T), Jag1-CTF-HA (pEF6/V5-His) and JICD-HA (pEF6/V5-His) HA-tagged expression plasmids were provided by Matthew LaVoie (LaVoie and Selkoe, 2003 (link)) and verified using Sanger DNA sequencing. Each plasmid was transfected into B3 cells using the Fugene6 (Promega, Cat#:E2691) protocol at a 3:1 volume ratio of Fugene6 transfection reagent relative to that of the DNA. For immunocytochemistry experiments, 2.0×10⁵ to 2.5×10⁵ B3 cells were plated per well of a 2-well chamber slide (Thermo Fisher Scientific, Cat#:177429) and transfected with 40 ng of plasmid after 24 h (∼60–70% confluency). In experiments generating material for western blot analysis, 1.0×10⁶ to 1.5×10⁶ B3 cells were plated onto a 100 mm tissue culture plate and transfected with 5 µg of plasmid after 24 h (60–70% confluency). Cells remained in transfection mix media for 48 h at 37°C and 5% CO₂ before either fixing for staining protocol or harvesting for protein extraction via cell-scraping in cold PBS plus cOmplete mini protease inhibitor tablet (Sigma-Aldrich, Cat#:11836153001), spinning briefly, and snap-freezing the cell pellet.

Release of cytochrome c from the mitochondria was measured by immunoblotting as previously described [31 (link)]. Cells were incubated with or without 100 nM crizotinib, 50uM chloroquine, or 100 nM crizotinib + 50 uM chloroquine for 6 hours. The cells were then obtained by scraping followed by gentle centrifugation at 1700rpm for 3 minutes. The pellets were then washed with cold PBS and re-spun for 3 minutes. Next the cells were lysed in an ice-cold buffer containing 250 mM Sucrose, 1 mM EDTA, 25 mM Tris, pH 6.8, 0.05% IGEPAL and a Complete Mini protease inhibitor tablet (Sigma-Aldrich) until the cells outer membrane was compromised as determined by the trypan blue exclusion assay. The cells were then centrifuged at 14,000 rpm for 5 min at 4 °C and the supernatant, containing the cytosolic fraction, was transferred to new tubes. The pellet containing the mitochondrial fraction was then suspended in lysis buffer, and cytochrome c was measured in each fraction by immunoblotting.