4 pmol of siRNA for 20 min at room temperature and the mixture was added drop-wise to wells. Reduced expression of target proteins was confirmed by western blot analysis of cell read me lysates. Preparation of cell lysates Cells (1��107) were harvested, washed three times in 20 mM iodoacetamide (with centrifugation for 5 min at 450 x g each time), and resuspended in cell lysis buffer (150 mM NaCl, 5 mM EDTA, 20 mM Tris-HCl pH 7.5, 0.5% Triton X-100). Resuspended cells were incubated on ice for 1 h with occasional mixing, and then stored at ?80��C overnight. The following day, the cell lysates were thawed on ice, and then centrifuged at top speed in a desktop microcentrifuge for 30 min at 4��C. The supernatants were transferred to new tubes and stored at ?80��C.

Aliquots of lysates were mixed with 5X sodium dodecyl sulfate loading dye (250 mM Tris-HCl pH 6.8, 10% w/v sodium dodecyl sulfate, 30% v/v glycerol, 5% v/v ��-mercaptoethanol, 0.02% w/v bromophenol blue) and boiled for 5 min prior to gel loading. Western blot analysis Cell lysate samples were boiled for 5 min and then loaded on 4�C20% Tris-glycine pre-cast gels (Invitrogen). Electrophoresis was performed at 125 V at room temperature. The proteins were transferred at 30 V for 2 h at room temperature to polyvinylidene difluoride Immobilon-P membranes (Millipore, Billerica, MA, USA). After overnight blocking in a 5% w/v solution of nonfat dry milk, the membranes were incubated with primary antibodies (diluted with 5% nonfat dry milk).

Incubation with anti-actin antibody (1:2,000) was for 1 h and incubation with anti-full-length APLP2 antibody (1:1,000), anti-APLP2 C-terminus antibody (1:1,000) or anti-APP C-terminus antibody (1:1,000) was for 1.5 h. After primary antibody incubation, 3 washes with 0.05% Tween-20 in phosphate-buffered saline (PBS) for 10 min were performed. The membranes were subsequently incubated for 1 h in secondary antibodies, diluted 1:10,000 in 0.05% Tween-20 in PBS, and washed 3 times for 10 min with 0.3% Tween-20 in PBS. For protein visualization, the membranes were incubated for 1 min in Pierce ECL Western Blotting Substrate, or (for anti-APLP2 C-terminus) in Pierce SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) and exposed to Kodak BioMax MR film (Rochester, NY, USA).

For densitometry of protein bands, quantification was done with the Molecular Imager ChemiDoc XRS system with Quantity One 1-D analysis software (BioRad, Hercules, CA, USA). Assessment of cellular growth Growth of cells was assessed using thiazol blue (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium Dacomitinib bromide, MTT) purchased from Sigma (St. Louis, MO, USA), by the MTT assay. Briefly, cells were seeded in 6-well plates at 1��105 cells per well 24 h prior to the start of any cell culture treatments. The addition of ��-secretase inhibitors to S2-013 or hTERT-HPNE cells signified the start of the time course.