Vicia villosa agglutinin vva

ELISA plates (MaxiSorp 96-well immunoplates; Thermo Fisher Scientific) were coated with the F(ab′)₂ fragment of 5 µg/mL goat anti-human serum IgA (50 µL/well, Jackson ImmunoResearch Labs, West Grove, PA) in PBS and then incubated at room temperature for 1 h. The plates were then blocked with 1% BSA in PBS and incubated with 50 µL deglycosylated IgA1 (5 µg/mL) at 4°C overnight. Subsequently, the plates were washed with PBS-T and incubated with each biotinylated lectin: Helix pomatia agglutinin (HPA, 5 µg/mL; EY Laboratories, San Mateo, CA), Vicia villosa agglutinin (VVA, 5 µg/mL; Vector Laboratories), peanut agglutinin (PNA, 2 µg/mL; Vector Laboratories, Burlingame, CA), and WFA (5 µg/mL; Vector Laboratories) at room temperature for 2 h. After washing, the plates were incubated with HRP-conjugated streptavidin at room temperature for 1 h. A 3,3′,5,5′-tetramethylbenzidine substrate solution (50 µL/well, Thermo Fisher Scientific) was added to the plates followed by incubation at room temperature until signal development. The reaction was stopped with 2 M sulfuric acid (50 µL), and the optical density was measured at 450 nm with a Wallac 1420 ARVO SX Multilabel Counter (Perkin Elmer).

Antibodies against total integrin β1 and activated integrin β1 (HUTS-21) were purchased from BD Biosciences (Lincoln Park, NJ). Integrin β1 blocking antibody (P4C10) was purchased from Millipore (Billerica, MA). Antibodies against C1GALT1, GAPDH, and focal adhesion kinase (FAK) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against phospho (p)-FAK was purchased from Cell Signaling Technology Inc. (Beverly, MA). Antibody against actin was purchased from GeneTex Inc. (Irvine, CA). Vicia villosa agglutinin (VVA) and peanut agglutinin (PNA) lectins were purchased from Vector Laboratories (Burlingame, CA). Human collagen IV, human fibronectin, murine laminin, bovine serum albumin (BSA), and protein de-glycosylation kit were purchased from Sigma (St Louis, MO).