Blue

For electrophoresis, equal amounts of total protein within each fraction were loaded onto a polyacrylamide gel. Proteins were dissolved in sample buffer (10% glycerol, 5% β-mercaptoethanol, 3% SDS, 0.0625 M Tris-HCl (pH 6.8), 0.01% bromophenol blue) and heated for 10 minutes at 100°C. Samples were resolved by one-dimensional SDS-PAGE as described by Laemmli (1970) using a 12% gel and the fractioned proteins were electroblotted onto a PVDF membranes (Millipore, 0,45 µm pore size) [44] (link). After blocking the membrane with 3% milk in PBS containing 0.1% Tween 20 (PBS-T), membranes were incubated with polyclonal antibodies against claudin-1 (1∶2000) (Invitrogen), phosphothreonine and phosphoserine (1∶2000) (Abcam), washed in PBS-T and further incubated with HRP-conjugated secondary antibodies (1∶4000) (Biorad). A liquid substrate system for membranes TM-blue (Sigma) was used to detect the enzymatic activity of the secondary antibody. The density ratio of the specific bands was quantified using ImageJ (National Institutes of Health, Bethesda, MD). Representative blots from multiple experiments (minimum of three) are shown in the figures. Blots were digitally contrasted to preserved relative intensity of specific claudin-1 bands.

In vitro-polarized Th17 or Th1 cells or ex vivo CD4 T cells were incubated with only the indicated recombinant cytokines at the following concentrations: IL-1β (10 ng/ml), IL-6 (20 ng/ml), IL-18 (50 ng/ml), IL-23 (10 ng/ml), TGFβ (5 ng/ml) and IL-12 (10 ng/ml), all purchased from R&D Systems. Control cultures were left without additional cytokines or plated in wells coated with anti-CD3 and supplemented with soluble anti-CD28 (5 μg/ml). Supernatants were collected after 48 hours and the production of IL-17A, IL-17F and IFNγ were detected by color development (TM-Blue; Sigma) of HRP-avidin substrate (Vector) following capture by antibodies directed against mouse IL-17A, IL-17F or IFNγ and detection by biotinylated anti-mouse IL-17A, biotinylated anti-mouse IL-17F, or biotinylated anti-mouse IFNγ, respectively (all purchased from BD Biosciences, IL-17F R&D Systems). ELISA was performed as previously described [10 (link)]. IL-22 ELISA was performed according to the manufacture's instructions (R&D Systems). The amounts of cytokine were determined from standard curves established with serial dilutions of recombinant murine IL-17A, IL-17F, IL-22 or IFNγ (R&D Systems).