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Fig. 7 | Molecular Medicine

Fig. 7

From: The Cytoplasmic Domain of Alzheimer’s Amyloid Precursor Protein Is Phosphorylated at Thr654, Ser655, and Thr668 in Adult Rat Brain and Cultured Cells

Fig. 7

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.

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