Immunoblot Analysis of Amyloid Precursor Proteins

Immunoblot analyses were performed as previously described (18 (link)). The following antibodies were used: anti-APP (Millipore, MAB348, 1:1000), anti–APP-CTFs (Abcam, ab32136, 1:1000), anti–amyloid β (6E10) (against the N-terminus of human Aβ; BioLegend, 803001, 1:1000), anti-sAPPβ (BioLegend, 813401, 1:1000), anti–PS1-CTF (Sigma-Aldrich, P5110, 1:500), anti-USP25 (Abcam, ab187156, 1:1000), anti-ubiquitin (Santa Cruz Biotechnology, sc-8017, 1:500), anti-GOLPH4 (Abcam, ab57271, 1:1000), anti-Na⁺/K⁺ ATPase (Santa Cruz Biotechnology, sc-21712, 1:500), anti-myc (Thermo Fisher Scientific, 132500, 1:1000), anti-HA (Sigma-Aldrich, H6908, 1:1000), anti-Flag (Sigma-Aldrich, F7425, 1:1000), and HRP-conjugated secondary antibodies (Thermo Fisher Scientific, 31430 or 31460; 1:3,000). A previously described anti-PS1-NTF rabbit polyclonal antibody AB14 (1:1000; ref. 40 (link)) and a previously described anti-BACE1 mouse monoclonal antibody 3D5 (1:1000; ref. 41 (link)) were used.

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Zheng Q., Song B., Li G., Cai F., Wu M., Zhao Y., Jiang L., Guo T., Shen M., Hou H., Zhou Y., Zhao Y., Di A., Zhang L., Zeng F., Zhang X.F., Luo H., Zhang X., Zhang H., Zeng Z., Huang T.Y., Dong C., Qing H., Zhang Y., Zhang Q., Wang X., Wu Y., Xu H., Song W, & Wang X. (2022). USP25 inhibition ameliorates Alzheimer’s pathology through the regulation of APP processing and Aβ generation. The Journal of Clinical Investigation, 132(5), e152170.

Publication 2022

MAB348 ab32136 sc-8017 anti-Na+/K+ ATPase H6908 F7425

Amyloid Anti antibody Antibodies Bace1 Human Immunoblot analyses Monoclonal antibody Mouse Na k atpase Rabbit Ubiquitin

Corresponding Organization : Jinzhou Kangning Hospital

Other organizations : Discovery Institute, Sanford Burnham Prebys Medical Discovery Institute, Xiamen University, Tsinghua University, Beijing Institute of Technology

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Variable analysis

independent variables

Antibodies used for immunoblot analyses: anti-APP, anti–APP-CTFs, anti–amyloid β (6E10), anti-sAPPβ, anti–PS1-CTF, anti-USP25, anti-ubiquitin, anti-GOLPH4, anti-Na+/K+ ATPase, anti-myc, anti-HA, anti-Flag
Previously described anti-PS1-NTF and anti-BACE1 antibodies

dependent variables

Protein expression levels and modifications as detected by the immunoblot analyses

control variables

Experimental conditions and procedures as previously described (18)

controls

Positive controls: None explicitly mentioned
Negative controls: None explicitly mentioned

Annotations

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