High Fat Diet Enhances β-Site Cleavage of Amyloid Precursor Protein (APP) via Promoting β-Site APP Cleaving Enzyme 1/Adaptor Protein 2/Clathrin Complex Formation
Fig 1
HFD promoted BACE1/clathrin complex formation in APP transgenic mice.
(A) Equal amounts of brain samples from 7–8 months age of the control APP mice and APP-HFD mice (fed with HFD for 20 weeks) were immunoprecipitated by an anti-BACE1 antibody, and then SDS-PAGE was conducted. As a negative control, samples were immunoprecipitated by a normal mouse IgG. To visualize proteins, the gels were stained with silver nitrate. These proteomics assays were repeated twice using different four brain samples from the control APP mice as well as four samples from APP-HFD mice. The band density around 180 kDa was significantly higher in APP-HFD mice than that in the control APP mice. The protein bands were excised and subjected to in gel trypsinization, and molecular mass analysis of the tryptic peptides indicated that the band was clathrin heavy chain. (B) Equal amounts of brain samples from 7–8 months age of the control APP mice and APP-HFD mice (fed with HFD for 20 weeks) were immunoprecipitated by a monoclonal anti-BACE1 antibody (2nd row) and then they were immunoblotted by an anti-clathrin antibody or by an anti-BACE1 antibody. As a negative control, samples were immunoprecipitated by a normal mouse IgG (top row). These immunoprecipitation assays were repeated twice using different four brain samples from the control APP mice as well as four samples from APP-HFD mice. HFD promoted the BACE1/clathrin complex formation in APP transgenic mice. (C) Immunoblotting analysis using an anti-clathrin antibody. Statistical analysis is shown in the bottom panel. The band density of the clathrin heavy chain was normalized by that of β-actin. The band density of the control APP mice was regarded as 100% and that of APP-HFD mice was relatively indicated. The band density of clathrin in APP-HFD mice (fed with HFD for 20 weeks) was the same as that in the control APP mice. n.s. indicates that there was no statistical significance.