Er stress antibody kit

Cells were harvested by scraping, before lysis in RIPA buffer (BioBasic) and clarification by centrifugation. Total cell lysates (40 μg) were separated using SDS-PAGE (20%, w/v) and transferred to a 0.2 um PVDF membrane with the Bio-Rad Trans-Blot Turbo Transfer System. The membrane was blocked for one hour with 5% skim milk in TBST buffer, followed by overnight incubation with the primary antibody in TBST at 4° Celsius. The membrane was washed three times with TBST before incubation with a species-specific horseradish peroxidase-linked secondary antibody for one hour at room temperature. Protein levels were determined using the Enhanced Chemiluinescence System (GE Healthcare) scanned on the BioRad Gel-Doc XRSystem (Bio-Rad). Antibodies used: anti-EroI, -calnexin, -PDI, -PERK and -BiP antibodies were obtained as part of the ER Stress Antibody Kit (Cell Signaling, cat. 9956 T), anti-EDEM1 (Abcam, cat. ab200645), anti-Fbx06 (Abcam, cat. ab57058), anti-human CFTR NBD2-specific (amino acids 1204–1211) murine monoclonal antibody 596 (1:30,000, University of North Carolina at Chapel Hill, Chapel Hill, NC, Courtesy Cystic Fibrosis Foundation Therapeutics){CHIBAFALEK:1999ey}. Secondary antibodies included Goat Anti-Mouse IgG (H + L)-HRP (Biorad cat. 1721011) and Goat Anti-Rabbit IgG (H + L)-HRP (Biorad cat. 1721019).