Polyvinylidene difluoride transfer membrane

Protein lysates (6.5–9 μg for cells and 25 μg for small intestine mucosal scrapings) in 1X SDS/β-mercapthoethanol buffer were resolved on a 4–20% TGS stain free gel (BioRad, Hercules, CA) and electrotransferred onto a polyvinylidene difluoride transfer membrane (Thermo Fisher Scientific). Western blot analysis was performed using rabbit anti-LC3A/B (1:1000; Cell Signaling Technology #4108, Danvers, MA), rabbit anti-SQSTM1/p62 (1:1000; Cell Signaling Technology #5114) or rabbit anti-GAPDH (1:1000; Cell Signaling Technology #5174) and horseradish peroxidase-conjugated goat anti-rabbit (1:2000; Cell Signaling Technology #7074) antibodies. Protein bands were visualized by chemiluminescence using a ChemiDocTouch Imaging System (BioRad). Bands were quantified using the ImageLab software Version 5.2.1.

Polyclonal antibody against LC3B and phosphorylated antibodies were purchased from Cell Signaling Technology (Danvers, MA). Polyclonal antibody against PKCε and monoclonal antibody against p62 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Monoclonal antibody against PARP was obtained from BD Pharmingen (San Diego, CA, USA). Monoclonal antibodies against actin and tubulin, and chloroquine diphosphate salt were purchased from Sigma (St. Louis, MO, USA). Bafilomycin A1 and rapamycin were obtained from LC Laboratories (Woburn, MA, USA). Human recombinant TNFα was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Horseradish-peroxidase-conjugated donkey anti-rabbit and goat anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Polyvinylidene difluoride transfer membrane was obtained from Thermo Fisher Scientific (Waltham, MA, USA) and the enhanced chemiluminescence detection kit was from Perkin-Elmer (Shelton, CT, USA). Protease inhibitor and phosphatase inhibitor cocktails were purchased from Calbiochem/EMD-Millipore (Bedford, MA, USA). Control non-targeting and target-specific siGENOME SMARTpool siRNAs were obtained from Dharmacon (Lafayette, CO, USA). Lipofectamine RNAiMax transfection reagent was obtained from Invitrogen (Carlsbad, CA, USA).

Proteins from cell lysates (20 µg of total proteins) or VLPs (volume to volume) were loaded and separated on 8% and 12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride transfer membrane (Thermo Fisher). Immunoblotting was performed by using the corresponding antibodies. Horse Radish Peroxidase signals were revealed by using Amersham ECL Prime (Sigma Aldrich). Images were acquired using the Chemidoc Imaging system (Bio-Rad). Each band intensity on the immunoblot was quantified using the ImageJ software. Quantification was established by the following formula for each viral protein: VLP/(VLP + cell lysate) relative to GAPDH as a loading control.