Anti interleukin il 6

Colonic tissues were lysed with RIPA buffer (Invitrogen Life Technologies, Carlsbad, CA, United States) and centrifuged at 13,000 rpm for 15 min at 4°C. Protein concentration was determined using a Bio-Rad protein assay kit (Hercules, CA, United States). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose blotting membrane (GE Healthcare United Kingdom Ltd. Little Chalfont, United Kingdom). The blots were incubated with anti-tumor necrosis factor (TNF)-α, anti-cleaved caspase-3 (all from Cell Signaling Technology Inc. Beverly, MA, United States), anti-interleukin (IL)-6, anti-IL-10, anti-cyclooxygenase (COX)-2, anti-Bax (all from Abcam, Cambridge, United Kingdom), anti-IL-1β, anti-occludin, anti-ZO-1, anti-claudin-2, anti-p53, anti-p21, or anti-β-actin (all from Santa Cruz Biotechnology) primary antibodies, and immersed in 5% bovine serum albumin overnight at 4°C. The blots were washed three times with PBS containing 0.05% (v/v) Tween 20 and incubated with secondary antibodies for 1 h at RT. The membranes were then washed three times with PBS and visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific Inc. Rochford, IL, United States).

BAC was purchased from Sigma (Sigma-Aldrich, St. Louis, MO). Preserved (0.01% BAC) 0.1% FML was purchased from Alcon (Flucon; Alcon Laboratories, Fort Worth, TX), and unpreserved 0.1% FML, FML powder, and original solvent were obtained from Hanlim (Fumelone eye drops 0.6 mL; Hanlim Pharm. Co., Seoul, South Korea). Antitumor necrosis factor (TNF)-α, antiinterleukin (IL)-6, and antihuman leukocyte antigen (HLA)-DR antibodies were purchased from Abcam (Cambridge, MA, USA). 4′,6-diamidino-2-phenylindole (DAPI) and ProLong Gold were purchased from Invitrogen (Carlsbad, CA). An in situ cell death detection kit for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was purchased from Roche (In Situ Cell Death Detection Kit, Fluorescein; Roche Diagnostics, Indianapolis, IN).

BAC and lissamine green B were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Anti-tumor necrosis factor (TNF)-α, anti-interleukin (IL)-6, and anti-IL-1β were purchased from Abcam (Cambridge, MA, USA). VECTASHIELD^® Mounting Medium with DAPI was purchased from Vector Laboratories (Burlingame, CA, USA). An in situ cell death detection kit for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was purchased from Roche (In Situ Cell Death Detection Kit, Fluorescein; Roche Diagnostics, Indianapolis, IN, USA). Anti-β-actin was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). NF-κB and p-NF-κB were purchased from Cell Signaling Technology (Danvers, MA, USA). CsA and FML were obtained from Hanlim (Tsporin eye drops 0.05% CsA; Fumelone eye drops 0.6 mL; Hanlim Pharm. Co., Seoul, South Korea).