Fig. 7From: The Cytoplasmic Domain of Alzheimer’s Amyloid Precursor Protein Is Phosphorylated at Thr654, Ser655, and Thr668 in Adult Rat Brain and Cultured Cells Phosphorylation of APP in adult rat brain (a) APP was immunoprecipitated from adult rat brain lysates (5 mg total protein) using G-369, pAbT654, or pAbS655 antibodies, as indicated. Samples were subjected to SDS-PAGE and immunoblotting as described above. Membrane strips were probed with either G-369, anti-KPI domain (56,1) or pAbT688 antibodies, as indicated. The arrows indicate the positions of mature and immature APP isoforms (see text). Lanes 1 and 2 represent different exposures of the same sample probed with G-369 antibody. (b) and (c) APP was immunoprecipitated from adult rat brain (7 mg total protein) using either G-369 or pAbT688 antibodies as indicated. Samples were subjected to SDS-PAGE and immunoblotting as described above. Membrane strips were probed with either G-369 or pAbT688 antibodies as indicated. Antigen +: Prior to immunoblotting, pAbT688 antibody was preincubated with APP695[665–673]P-Thr668, Cys673 (10 µM). In some cases prior to immunoblotting, immunoprecipitates were incubated with bacterial alkaline phosphatase (40 unit) (BAP+) or without any addition (BAP−) in a buffer containing 0.5 M Tris-HCl (pH 7.9), 12.5 mM MgCl2, 8.3% (v/v) glycerol, 200 µg/ml (w/v) pepstatin A, 200 µg/ml (w/v) leupeptin, and 200 µg/ml (w/v) chymostatin. The arrows indicate the positions of immature isoforms of APP. (d) APP was immunoprecipitated from adult rat brain (5 mg total protein) with G-369 or G-530 (anti-rat Aβ1–16) antibodies as indicated. Samples were subjected to SDS-PAGE and immunoblotting as described above. Membranes were probed with G-369 or pAbT668 antibodies as indicated. The arrows indicate immature APP isoforms.Back to article page