Sc 8017

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.

Proteins were separated by SDS–PAGE and transferred to nitrocellulose membranes (GE Healthcare) using wet blot transfer. Total protein was stained using Ponceau S and membranes were blocked and incubated with antibodies either in 5% milk or 5% BSA in TBS (150 mM NaCl and 20 mM Tris pH 8.0). Washes were performed in TBS-T (TBS and 0.1% Tween20). After incubation with horesradish peroxidase-conjugated secondary antibodies, membranes were developed using chemiluminescence reagents ImmunoCruz (Santa Cruz) or TMA-6 (Lumigen). The following commercial primary antibodies were used: α-HA (16B12, Covance MMS-101, 1:1,000), α-GFP (B-2, Santa Cruz sc-9996, 1:1,000), α-FLAG (M2, Sigma F3165, 1:5,000), α-Ub (P4D1, Santa Cruz sc-8017, 1:1,000), α-TRIM56 (Abcam ab154862, 1:10,000), α-TRIM65 (Sigma HPA021578, 1:500), α-Tubulin (Sigma T9026, 1:5,000) and α-Vinculin (Sigma hVIN-1, 1:2,000). Uncropped scans of developed membranes are provided in Supplementary Figs 6,7,8 and 9 as part of the Supplementary Information.