Syvn1

Total protein was separated from cells by SDS-PAGE and then was transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Then membranes were blocked in TBS with 5% no-fat milk and incubated with primary antibodies, SYVN1, PCNA, cleaved-caspase 3, beclin1, LC3 and GAPDH (1:2000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The membranes were incubated with HRP-conjugated secondary antibodies (1:1000, Santa Cruz Biotech, Santa Cruz, CA). The blots were detected using Applied the PierceTM ECL western blotting substrate (ThermoFisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocols.

The mouse-specific membralin antibody used in this study was previously custom-made and now is available from Millipore (ABN1661, 1:1000). The PS1-NTF antibody used in this study was described previously (Rabbit polyclonal antibody, 1:1000)^{35 (link)}. Other antibodies were acquired from commercial sources as follows: c-Myc (Sigma, C2956, 1:1000), HA (Cell Signaling Technology, 3724 s, 1:1000), EMC3 (Santa Cruz, sc-365903, 1:1000), SYVN1 (Sigma, H7790, 1:500), SYVN1 (Santa Cruz biotechnology, sc-293484, 1:1000), AMFR (Cell Signaling Technology, 9590 s, 1:1000), AMFR (Santa Cruz Biotechnology, sc-166358, 1:1000), VCP (Thermo Fisher Scientific, MA3-004, 1:1000), VCP (Cell Signaling Technology, 2648 s, 1:1000), PS1-CTF (Millipore, 5232, 1:2000), human membralin (Sigma, HPA042669, 1:500), nicastrin (Cell Signaling Technology, 9447 s, 1:1000), nicastrin (Millipore, MAB5556, 1:1000), β-actin (Sigma, 5441, 1:10000), NICD (Cell Signaling Technology, 4147 s, 1:1000), PEN2 (Cell Signaling Technology, 8598 s, 1:1000), PSD95 (Cell Signaling Technology, 3450 s, 1:1000), active (cleaved) caspase3 (Cell Signaling Technology, 9662 s, 1:100), MOAB2 (Aβ40/42_, Biosensis, M-1586-100, 1:500), rabbit IgG control (Cell Signaling Technology, 2729, 1 μg used as IP control).