Horseradish peroxidase labeled anti mouse

The cells were tested in the absence or presence of TLR2 ligands after having reached 80% confluency. The cells were collected after treatment with PGN and P3C after 21 and 25 h, respectively. Cells were detached by means of a cell scraper and total-cell lysates were prepared for analysis. The denatured proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred electrophoretically onto a nitro cellulose membrane (Biorad) (20V, 20mins, 1Amp). The membranes were blocked using 5% non-fat milk and 0.05% Tween 20 in Tris-buffered saline at room temperature for 1 h and immunoblotted overnight with anti-phospho-PKCζ (Santa Cruz Biotechnology, Heidelberg, Germany), anti-claudin-1 (Invitrogen), anti-ZO-1, anti-occludin, and anti-actin antibodies (Sigma-Aldrich) and anti-cingulin³⁸ (link) at the dilutions recommended by the manufacturers at 4 °C. The membranes were incubated for 1h at room temperature with horse-radish peroxidase labeled anti-mouse (Sigma-Aldrich) or anti-rabbit IgG (Merck KGaA). Detection was performed using enhanced chemiluminescent western blotting system (Applichem, Darmstadt, Germany). To confirm equal loading, immunoblots were probed with anti-actin or anti-pkc ζ (Santa Cruz Biotechnology). All experiments were repeated at least 3 times; representative blots are shown for each experiment.

Cells were lysed for 30 min in Triton buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% sodium deoxycholate, and 0.1% SDS) supplemented with protease and phosphatase inhibitors (1 mM PMSF, 2 mM sodium pyrophosphate, 2 mM sodium betaglycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Lysates were cleared by centrifugation at 15,000 × g at 4°C for 15 min, and protein concentrations were determined by the Bradford method. Fifty micrograms of protein was separated by SDS-PAGE and transferred onto Immobilon-P membranes. Proteins were detected with special antibodies. Antibody binding was detected with horseradish-peroxidase-labeled anti-mouse (Sigma) or anti-rabbit (Cell Signaling) antibodies, and chemiluminescence was detected with a LAS4000 device (Fuji). Equal protein loading was confirmed with antibodies against β-actin (Transgen). Detailed information on antibodies is shown in Table S5.