The C-terminal fragment from the key cleavage solution from the WA mutant was only faintly detected, suggesting that the WA mutant could possibly not be cleaved

Materials and Techniques Plasmids The building of pcDNA3.1-hAlca1, pcDNA3.1-FLAGhAlca1, pcDNA3.1-Alca1-FLAG, pcDNA3.1-FLAG-hAPP, pcDNA3.1-hAPP-FLAG, pEGFP-KIF5C, and pcDNA3.1FLAG-KLC1 was previously described. Plasmids expressing point mutations of Alca1 resulting within a tryptophan-to-alanine substitution were prepared by replacing the target sequence with all the corresponding sequence carrying the indicated mutation, generated by PCR. Plasmids expressing Nterminal human placental alkaline phosphatase fusion proteins were generated by inserting the indicated PCR-amplified fragments in to the XhoI or XbaI website of AP-tag5. Substitution mutants with the Alca primary cleavage internet site were generated by the megaprimer PCR technique. Plasmids expressing primary cleavage web site substitution mutants or the AP-fusion form of Alca with WA mutations had been prepared by exchanging the C-terminal region of each and every Alca to that on the WA mutant. The resultant plasmids have been verified by sequencing. Antibodies and western blotting The following antibodies were employed. Anti-FLAG mouse monoclonal antibody, antia-tubulin mouse monoclonal antibody, rabbit anti-GFP, anti-APP, guinea pig anti-Alca, and rabbit anti-Alca. Western blotting was performed as described. for 30 min on ice. The reaction was stopped by washing the cells with PBS containing 50 mM glycine. The surface biotinylated cells had been lysed in 400 ml of RIPA buffer as well as the biotinylated proteins have been bound to immobilized NeutrAvidin agarose. The bound proteins were washed and subjected to SDS-PAGE for immunoblotting. The absence of a-tubulin from the NeutrAvidin-bound fraction was utilised to confirm that the intracellular proteins have been not biotinylated. FLAG-tagged proteins were detected on the surface of reside cells as follows. CAD cells have been transfected with plasmids expressing the indicated FLAG-tagged proteins and cultured on poly-Dlysine-coated 8-well cover-glass chamber slides. The cells have been washed with ice-cold PBS and incubated with 1 mg/ml anti-FLAG mouse monoclonal antibodies in 1% heat-inactivated goat serum/ PBS at 4uC for 30 min. Right after being washed with ice-cold PBS three times, the cells had been fixed with 4% PFA/PBS at 4uC for 30 min. The fixed cells were washed with PBS and further incubated with Cy3-conjugated donkey Nonetheless, no important distinction was observed inside the volume of extracellularly liberated AP activity in between the wild type- and WA mutant-expressing cells anti-mouse IgG in 1% heat-inactivated goat serum/PBS containing 5 mg/ml Hoechst 33342 to visualize the bound anti-FLAG antibodies and cell nuclei. The cells had been washed with PBS, and their fluorescent images were serially collected having a BZ-9000 microscope utilizing exactly the same conditions for each and every specimen. Following collecting the photos, the cells have been incubated in 0.1% Triton X100/PBS for 5 min at room temperature. The resultant permeabilized cells had been incubated with 1 mg/ml anti-FLAG mouse monoclonal antibodies in 1% heat-inactivated goat serum/PBS at 4uC for 30 min. Right after washing the cells with PBS, the bound anti-FLAG antibodies were visualized using the secondary antibodies. The resultant fluorescent photos in the permeabilized cells had been taken with the BZ-9000 microscope. Representative images on the very same cells before and soon after permeabilization are shown inside the figures. Analyses of alkaline phosphatase activity and p3-Alca released in to the cell culture medium Plasmids expre