Anti bag1

For tissue collection, mice were deep anesthetized, decapitated and each hippocampus was rapidly dissected and stored at −80℃ until use. Immunoblotting was performed as described previously [3 (link)]. In brief, the hippocampus was denatured in lysis buffer and the protein concentration was determined by BCA method (Bio-rad, CA, USA). Then it was resolved by SDS-PAGE and transferred to the PVDF membrane. Primary antibodies (anti-BAG1; sc-377454, anti-beta actin; sc−517582, Santa Cruz Biotech. Dallas TX) were diluted to a 1:1000 concentration ratio and applied overnight at 4℃. After an hour of incubation in 1:1000 diluted secondary antibody, bands were visualized by ECL solution (Thermo Fisher Scientific, MA) and quantified with a chemiluminiscence detector (Davinch Chemi imaging system, CellTagen, Korea) and the ImageJ image analysis software (NIH, USA).

Cells were harvested from 100 mm dishes by rinsing twice with PBS and then lysed on the plate with 70 μl RIPA buffer (Thermo, USA), and 1× complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) on ice for 30 min. Cell lysates were centrifuged at 4°C at 12000 r/min for 20 min. The supernatant (protein) was retained; protein content was quantified. An aliquot of the total protein (30 μg) was loaded into each lane of a 12% sodium dodecyl sulfate polyacrylamide gel and then electrophoretically transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% nonfat milk in 0.01 mol/L TBS and 0.05% Tween-20 (TBST) at room temperature for 2 h before incubation with anti-BAG1, anti-BCL-2, anti-TP73 (dilution 1:1000; Santa Cruz, California, USA), anti-CASP1, anti-LTBR, or anti-CASP4 (dilution 1:1000; Abcam, Cambridge, MA, USA) antibodies at 4°C overnight, as appropriate. After three quick washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Proteintech, Wuhan, China) for 2 h at room temperature. Bands were visualized using ECL Western Blotting Substrate (Pierce, Rockford, USA).