Figure 6

p23 depletion affects the co-residence of APP and Mint3. A, N2a cells transfected with control or p23 siRNA were analyzed by immunofluorescence staining with a mAb against APP adaptor Mint3 and polyclonal p23, β-COP, giantin, or APP antibodies. Bar represents 10 μm. B, siRNA transfected N2aSwe cells were lysed using a ball-bearing homogenizer and fractionated by velocity sedimentation. Equal volume aliquots of fractions were analyzed by immunoblotting using antibodies against Mint3 and APP. C, Aliquots of fractions 6–12 were pooled and incubated with magnetic beads coated with mAb OKT8 (negative control) or mAb 9E10 (recognizes C-term myc tag in APP). Bound membranes and vesicles were solubilized in Laemmli buffer and analyzed by SDS-PAGE and Western blotting. D, Immunoisolation was performed using N2aSwe cells transfected with control or p23 siRNA and relative signal intensity of co-immunoisolated Mint3 relative to APP was quantified and graphically represented.