Rabbit anti claudin 3

Fullerenol, C60(OH)₂₄ was purchased from BuckyUSA (Houston, TX, USA) and dissolved in the vehicle solution composed of 0.9% NaCl. Fullerenol was diluted with cell culture medium to 1 (0.19 µM), 10 (1.9 µM) and 100 µg/mL (19 µM). IFN-γ was purchased from Miltenyi (Bergisch Gladbach, Germany). TNF-α was obtained from Peprotech (Hamburg, Germany). Mouse anti-alpha smooth muscle actin, rabbit anti-GFAP, rabbit anti-occludin and rabbit anti-VCAM-1 antibodies were acquired from Abcam (Cambridge, UK). Rabbit anti-caspase 3, cleaved caspase 3, erk1/2, phospho erk1/2 and phospho NFκB p65 were obtained from Cell Signaling (Leiden, The Netherlands). Goat anti-NFκB p65 and ICAM-1, and rabbit anti-claudin 5 were from Santa Cruz (Heidelberg, Germany). Rat anti-CD31 was obtained from Serotec (Puchheim, Germany). Rat anti-VE-cadherin was purchased from eBiosciences (Frankfurt, Germany). Rabbit anti-claudin 3 was obtained from Invitrogen (Karlsruhe, Germany). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Proteins were extracted from whole colonic tissue as described previously [24 (link)]. The protein extract (30 µg) was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% (w/v) milk in PBS-T buffer for 1 h at room temperature and then incubated with the following specific primary antibodies: Rabbit anti-ZO-1 (dilution; 1:500, Invitrogen, Camarillo, CA, USA), rabbit anti-occludin (dilution; 1:250, Invitrogen), rabbit anti-claudin 1 (dilution; 1:500, Invitrogen), rabbit anti-claudin 3 (dilution; 1:1000, Invitrogen), rabbit anti-claudin 4 (dilution; 1:250, Invitrogen), and rabbit anti-GAPDH (dilution; 1:1000, Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. After washing with PBS-T buffer, the membranes were incubated for 1 h with the corresponding horseradish-peroxidase-conjugated secondary antibodies (Cell Signaling Technology). The target proteins were detected using an enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). ImageJ software (NIH) was used for quantifying the intensities of the target bands. The staining intensity of GAPDH was set as the internal control. The values in individual tests were expressed as fold values of the target protein/GAPDH in the standard group.