Ripa buffer

Western blot analysis was performed as previously described with a slight modification [31 (link)]. Treated samples were lysed in radioimmunoprecipitation assay (RIPA) buffer (HaiGene, China) containing protease inhibitor cocktail and phosphatase inhibitors (Roche, Switzerland), separated by SDS-PAGE under reducing conditions, and transferred onto a PVDF membrane (Merck Millipore, USA). After blocking, the membranes were incubated with a primary antibody and then incubated with an appropriate IRDye-conjugated secondary antibody (Li-Cor Biosciences, Lincoln, NE). The membranes were scanned using an Odyssey instrument (Li-Cor Biosciences) according to the manufacturer’s instructions.

The total cellular samples were washed twice with PBS and lysed in RIPA buffer (HaiGene) supplemented with 1 mM PMSF (Sigma-Aldrich, St. Louis, MO). The concentration of total protein was determined using BCA Protein Assay Kit (Pierce, Rockford, IL). The total cellular protein extracts were separated by SDS–PAGE and transferred onto the nitrocellulose membrane (PALL, Washington, NY) in 20 mM Tris–HCl (pH 8.0) containing 150 mM glycine and 20% (v/v) methanol. The membrane was blocked with 5% nonfat dry milk in 1 × TBS containing 0.05% Tween 20 and incubated with primary antibody at 4 °C overnight. Antibodies against Akt (1:1000), phospho-Akt (Ser473) (1:500) were purchased from Cell Signaling Technology. Antibodies against ASK1 (1:500), phospho-ASK1 (Thr845) (1:500), p38 (1:1000), phospho-p38 (Tyr182) (1:500), β-Actin (1:1000) and secondary antibodies (1:5000) were all purchased from Santa Cruz (Weatherford, TX). Occludin antibody (1:1000) was obtained from Invitrogen, and ZO-1 antibody (1:1000) was obtained from Abcam (Cambridge, MA). The signals were developed with enhanced chemiluminescence reagent (HaiGene), and the digital images were captured using a LAS-4000 CCD camera system (Fuji film, Tokyo, Japan). The relative intensity of bands was analyzed by the software of Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD).

Western blotting analysis was conducted as previously described with minor modifications [31 (link)]. The treated samples were harvested using radioimmunoprecipitation assay (RIPA) buffer (HaiGene, Harbin, China) supplemented with a protease inhibitor cocktail and phosphatase inhibitors (Roche, Indianapolis, IN, USA). The samples were then separated by SDS-PAGE under reducing conditions. Subsequently, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Temecula, CA, USA). The membrane was blocked in 5 % (w/v) skim milk for 1.5 h and incubated with a primary antibody and subsequently treated with an appropriate IRDye-conjugated secondary antibody (Li-Cor Biosciences, Lincoln, NE, USA) for 1 h at room temperature. Finally, the membranes were scanned using an Odyssey instrument (LiCor Bio-Sciences, Lincoln, NE, USA) according to the manufacturer’s instructions.