Seeblue plus 2 protein markers

Discontinuous SDS-PAGE was carried out through a running gel containing 12 % (w/v) total acrylamide using NuPAGE pre-cast Bis-Tris gels and Seeblue Plus 2 protein markers (Invitrogen, Paisley, UK). Electrophoresed gels were stained using 0.05 % (w/v) Coomassie Brilliant Blue R250 (Fischer Scientific). For Western blots, separated proteins were transferred to polyvinylidine difluoride membrane (Roche) using a NuPAGE Blot module (Invitrogen) at 30 V for 1 h. The 9E10 anti-C-myc antibody was used to identify scFvs. For staining of Kit in Western blots, a mouse IgG1 anti human Kit antibody (E1, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was used.

NuPage^® 4–12% polyacrylamide gels, LDS sample buffer, See-Blue Plus2 protein markers, and TRIzol^® reagent were purchased from Invitrogen (Carlsbad, CA, USA); iScript™ cDNA synthesis Kit and SYBR™ green supermix from Bio-Rad Laboratories (Hercules, CA, USA), primers for real-time PCR from Integrated DNA Technologies (Coralville, IA, USA). Antibodies against Claudin-1 (which also recognizes Claudin-3) and -2 were purchased from ThermoFisher Scientific (Waltham, MA, USA). Goat polyclonal actin primary antibody, and horseradish peroxidase-conjugated secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and SuperSignal West Dura substrate from Pierce (Rockford, IL, USA). A mouse TH1/TH2 9-Plex assay kit was purchased from MesoScale Discovery (Gaithersburg, MD, USA). All other reagents used were commercially available and were of the highest quality available.