Tlr4 ab13556

Western blot was performed as previously described [6 ]. Briefly, equal amounts of protein cell extract (30 μg) were boiled in 5xSDS loading dye (50% glycerol, 0.5 M dithiothreitol, 350 mM SDS, 7.5 mM bromphenolblue, 250 mM TRIS, pH 6.8) for 5 min and subjected to 8%, 10% or 13% sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis. After protein transfer onto PVDF membranes (Immobilon-P, Millipore, Schwalbach, Germany) the membranes were blocked with 5% skim milk in Tween20/PBS and incubated in the recommended dilution of protein specific antibody (p-ERK1/2 #4370, p-IRAK4 #11972 and MyD88 #4283; all Cell Signaling Technology, Danvers, MA, USA, TLR2 #ab24192, TLR4 #ab13556, both Abcam plc, Cambridge, UK) overnight at 4°C. After incubation with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Freiburg, Germany), proteins were visualized using ECL Western blotting detection reagent (Western Lightning plus ECL, #NEL103001EA, PerkinElmer, Waltham, MA, USA) following the manufacturer´s instructions. Images were generated with Fusion Fx^® imaging system (PEQLAB Biotechnologie GmbH, Erlangen, Germany). For normalization, blots were re-probed with total ERK1/2 (#4695), total IRAK4 (#4363) and ß-Actin (#4967), all Cell Signaling Technology, Danvers, MA, USA.