Intercept tris buffered saline blocking buffer

Samples were separated by SDS-PAGE on 4% to 20% polyacrylamide gels and transferred to nitrocellulose blots using a Turbo Blot transfer system (Bio-Rad). Blots were blocked for 1 h at room temperature in Intercept Tris-buffered saline (TBS) Blocking Buffer (LI-COR), then incubated in primary antibody dissolved in TBS containing 0.05% Tween-20 (TBS-T) overnight at 4 °C. Blots were washed for 5 min 3× in TBS-T, then incubated in secondary antibody dissolved in Intercept T20 Antibody Diluent (LI-COR) for 1 h at room temperature. Blots were washed for 5 min 3× in TBS-T and imaged on an Odyssey FC Imaging System (LI-COR) at 700 and 800 nm wavelengths. Antibodies and dilutions used were as follows: ⍺-CHIP (1:2000, abcam #ab109103), ⍺-tau (1:1000, Santa Cruz Biotech #sc-1661060), IRDye 680RD goat anti-mouse secondary (1:10,000, LI-COR #926-68070), IRDye 800CW goat anti-rabbit secondary (1:10,000, LI-COR #926-32211).

24 h after transfection, cells were washed with phosphate-buffered saline (PBS) and lysed with RIPA (Radioimmunoprecipitation assay) buffer with protease inhibitors (Thermo Fisher) and DNAse I (NEB). The lysates or cell supernatants were mixed with western blot (WB) loading dye (NuPAGE™ LDS Sample Buffer) and reducing agent (NuPAGE™ Sample Reducing Agent), denatured by heating, and loaded onto polyacrylamide gels (Bolt™ Bis-Tris Plus Mini Protein Gels, 4‐12%). The proteins were transferred to a PVDF membrane using the BioRad Trans-Blot Turbo system. The membranes were blocked with Intercept Tris-buffered saline (TBS) Blocking Buffer (LI-COR), stained with anti-gE primary antibody (GeneTex), and a goat anti-mouse fluorescently labeled secondary antibody (Invitrogen). The membranes were imaged on a LI-COR Odyssey.