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Figure 5 | Molecular Neurodegeneration

Figure 5

From: Hyperpolarization-activated cyclic nucleotide gated channels: a potential molecular link between epileptic seizures and Aβ generation in Alzheimer’s disease

Figure 5

APP and HCN1 form a molecular complex in vivo and in vitro . (A) Co-immunoprecipitation of the HCN1-APP complex from the wild type and HCN1-/-murine cortex. Brain lysates were immunoprecipitated with an anti-HCN1 antibody. Immunocomplexes were detected by immunoblotting. (B) Co-immunoprecipitation of HCN1-APP, -X11 and -X11L complexes from the EC-rich cortex (see Additional file 1: Figure S2). Brain lysates were immunoprecipitated with an anti-HCN1 antibody. Immunocomplexes were detected by immunoblotting. (C–G) Co-immunoprecipitation of the HCN1-APP complex from N2a cells transiently overexpressing FLAG-APP and murine HCN1. (C–E), Analysis of FLAG-APP and murine HCN1, (F) C99-FLAG and murine HCN1, and (G) APPFL or APPΔcyt and murine HCN1 immunocomplexes. To standardize the amount of plasmid, empty vector (−) was added to yield 1.2 μg of plasmid in total. Cell lysates were immunoprecipitated with anti-FLAG M2 (C and F), anti-APP extracellular domain (LN27) (D), or anti-HCN1 (E and G) antibodies. Immunocomplexes were detected by immunoblotting. (H) Affinity purification of FLAG-sAPP secreted into the culture medium by N2a cells expressing FLAG-APP. The purification was performed using anti-FLAG M2 affinity beads. FLAG-sAPP were specifically detected by M2 and anti-APP extracellular domain (LN27) antibodies, and no contaminating bands were identified by CBB-staining. (I) Pull-down of HCN1 with affinity purified FLAG-sAPP prepared in (H). Lysates from wild type cells (−) and cells that transiently expressed HCN1 (+) were incubated with M2 affinity beads coupled with FLAG-tag or FLAG-sAPP. The complexes resulting from the pull-down assay were subjected to immunoblot analysis with anti-HCN1 antibody.

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