Zo 1 61 7300

The skin, corneal epithelium, lung tissue or cultured cells were extracted with cold lysis buffer comprising 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P‐40, 0.5% sodium deoxycholate, 0.1% SDS, and protease and phosphatase inhibitor cocktails. Equal amounts of protein extracts were subjected to electrophoresis on 8% or 10% SDS‐PAGE and then electrophoretically transferred to PVDF membrane. After blocking in 1% BSA for 1 hr, the membranes were incubated with primary antibodies ZO‐1 (61‐7300, 1:1000, Invitrogen), claudin‐1 (519,000, 1:1000, Invitrogen), Gli‐1 (sc‐20687, 1:500,Santa Cruz) and β‐actin (A3854, 1:10,000, Sigma‐Aldrich) overnight at 4°C. After three washes with Tris‐buffered saline with 0.05% Tween‐20 for 10 min. each, the membranes were incubated with HRP‐conjugated goat or rabbit anti‐mouse or rabbit IgG (1:10,000) for 1 hr. The results were visualized by enhanced chemiluminescence reagents and recorded with an imaging system (ChemiDoc XRS, Bio‐Rad, Hercules, CA, USA).

Intestinal tissues were homogenized and lyzed in radio-immunoprecipitation assay buffer, and protein concentrations were determined using a bicinchoninic acid protein quantitation kit. Lysate samples were fractionated by sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes, blocked with 5% non-fat milk for 1.5 h at room temperature, and incubated with primary antibodies overnight at 4°C. Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were applied for 1–2 h at room temperature, prior to detection with an enhanced chemiluminescence kit. Antibodies to anti p-p38MAPK (Thr180/Tyr182; 4511), anti-p-ERK1/2 (Thr202/Tyr204; 9101), anti-p-JNK (Thr183/Tyr185; 4668), anti-p38MAPK (9212), anti-ERK1/2 (4695), anti-JNK (9252), anti-TLR4 (14358), anti-CD11c (97585), and anti-F4/80 (30325) were obtained from Cell Signaling Technology (CST, Danfoss, MA, USA). Antibodies against zonula occludens-1 (ZO-1, 61-7300) and occludin (33-1500) were purchased from Invitrogen (USA). β-Actin antibody (C1313), goat anti-rabbit IgG/HRP (C2226), and goat anti-mouse IgG/HRP (C2225) were purchased from Applygen Technologies Inc. (Beijing, China). Protein levels were normalized to those of β-actin.