Calpain 1

Equal amounts of protein were loaded in each well of 4-20% Tris-glycine gels (Bio-Rad) as recently described [33 (link)]. After electrophoresis for 90 min at 125 V of constant voltage, the gel was blotted onto a nitrocellulose membrane by electrophoretic transfer at 90 V of constant voltage for 1.5 h. The membrane was washed, blocked with 5% blocking solution, and probed with various primary antibodies at 4°C overnight. Antibodies: Mitofilin (Polyclonal, Proteintech 10179-1-AP, 1 μg/ml, and Monoclonal, Invitrogen (2E4AD5, 45-6400) at 2 μg/mL for WB), Cyclophilin D (Novus Biologicals, 455900, 1 μg/ml), Calpain 10 (Santa Cruz, (C-20), sc-48454 1 μg/ml), Calpain 1 Santa Cruz, (D-11), sc-271313 1 μg/ml), Myc-Tag Antibody (Cell Signaling, #2272 1 μg/ml), VDAC1 (Santa Cruz (B6) sc-390996, 5 μg/ml), GAPDH (Cell Signaling, D4C6R #9716, 5 μg/ml). After washing, membranes were incubated for 1h at room temperature with the corresponding fluorophore-conjugated secondary antibodies (goat anti-rabbit Alexa 680, 20 ng/ml; goat anti-mouse IR Dye 800CW, 10 ng/ml). The total protein loading was measured by immersing the membrane in the ponceau S solution (CAS No.: 6226-79-5). After washing, bands were visualized using an infrared fluorescence system (Odyssey Imaging System, Li-COR Biosciences).

Panc 10.05 cells grown in light RPMI1640 media were lysed in M-PER buffer supplemented with protease inhibitor cocktail. The lysate was immunoprecipitated with either the pre- or post-vaccination purified antibodies using protein G beads. The immunoprecipitates were eluted by boiling in NUPAGE LDS sample buffer and resolved on a NuPAGE 4-12% Bis-Tris gel (Invitrogen). Proteins in the gel were transferred onto nitrocellulose membrane using a semi-dry apparatus (Invitrogen). The membrane was blocked in 5% bovine serum albumin (BSA, Invitrogen) in 0.1% Tween 20-PBS (PBS-T) buffer for 1 hour at room temperature and probed with the relevant primary antibody overnight at 4°C. Antibodies against galectin-3 (sc-19283), E3 ubiquitin protein ligase (sc-9561), Mesencephalic astrocyte-derived neurotrophic factor (sc-34560), Epidermal growth factor receptor kinase substrate 8-like protein 2 (sc-100722), Calpain-1 (sc-81171) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The membrane was incubated with the corresponding peroxidase conjugated secondary antibodies (Sigma) and then ECL Western Blotting Detection Reagents (GE Healthcare) was used for developing.