Newborn calf serum

F12 (Coon’s modification) medium and BFA (brefeldin A) were purchased from Sigma–Aldrich. BFA was stored at −20°C as a 10 mg/ml stock in DMSO. Lactacystin was stored as a 5 mM stock at −20°C. MG-132 (Cell Signaling Technology) was stored at −20°C as a 10 mM stock in DMSO. FBS and newborn calf serum were purchased from Gemini Bio-Products. TaqSelect DNA polymerase was purchased from Lucigen. High-speed plasmid mini kits were from MidSci. HRP (horseradish peroxidase)-conjugated secondary antibodies and Western Lightning ECL reagent were purchased from Sigma–Aldrich and PerkinElmer respectively. Alexa Fluor^®-conjugated secondary antibodies were purchased from Invitrogen. Monoclonal antibodies against the V5 epitope tag were from AbD Serotec or against mannosidase II were purchased from Covance. ProteoStat^® was purchased from Enzo Life Sciences and cells were stained according to the manufacturer’s instructions. LC3A/B antibodies were from Cell Signaling Technology. Anti-MAL2 polyclonal antibodies were generated and affinity-purified as described previously [2 (link)]. Polyclonal antibodies against haptoglobin, HA321, CE9, rat serum albumin, APN (aminopeptidase N) and lysosomal glycoprotein 120 were provided by Dr A. Hubbard (Johns Hopkins University School of Medicine, Baltimore, MD, U.S.A.).

Cells were maintained at 37°C in 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) with bovine serum and penicillin–streptomycin. Vero cells (BEI Resources) were supplemented with 10% newborn calf serum (GeminiBio), and Huh7, BHK-21, MEF, and HFF cells were supplemented with 10% fetal bovine serum (GeminiBio).