Tlr 4

Serial paraffin slices were studied immunohistochemically by standard methods. A panel of antibodies to CD3, CD 68 (CellMarque, rabbit monoclonal antibodies, dilution titer 1:1000), CD20 (CellMarque, rabbit monoclonal antibodies, dilution titer 1:500), perforin (CellMarque, mouse monoclonal antibodies, dilution titer 1:50), toll-like receptor type 4 (TLR-4, GeneTex, rabbit polyclonal antibodies, dilution titer 1:200), TLR-9 (GeneTex, rabbit polyclonal antibodies, dilution titer 1:50) was used.

We collected equal amounts of protein (20–40 μg) from the whole bladders of treatment groups described above and subjected them to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8–12%) and electrotransfer to a hydrophobic polyvinylidene difluoride (PVDF) membrane, as described previously [24 (link)]. The PVDF membranes were blocked and incubated overnight at 4°C with a primary antibody against pp65 NFκB (1 : 1000, BD PharMingen, San Diego, CA, USA), CD11b (1 : 500, Abcam, Cambridge, UK), SCF (1 : 1000, Santa Cruz Biotechnology, Inc., Santa. Cruz, CA, USA), muscarinic M3 (1 : 1000, US Biologicals, CO, USA) and TLR4 (1 : 1000, GeneTex, Irvine, CA, USA), TRPA1 (1 : 1000, Novus Biologicals, USA), or β-actin (1 : 1000, Millipore, CA, USA). After incubation of the samples with appropriately calculated secondary antibodies and developing with enhanced chemiluminescence (ECL) reagents, the Western blot on the membrane was visible on the X-ray films for quantification by imaging software (AlphaEaseFC, Alpha Innotech Corporation, USA).

SAC (purity 98.6%) (Figure 1A) was obtained from Chem Faces Pharmaceutical Company (Wuhan, China). Cisplatin, amifostine (AMF), EX-527 and other solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). BUN and CRE assay kits were supplied by HUMAN Diagnostics Worldwide (Wiesbaden, Germany). ELISA Max TM Set Deluxe kits, used to test IL-1β, IL-6 and TNF-α in mice, were obtained from BioLegend Inc. (San Diego, CA, USA). For Western blotting, primary antibodies that contrapose COX-2, p-JNK, catalase, SOD1, Sirt1, AMPK, GPx3 and TLR-4 were bought from GeneTex (San Antonio, TX, USA). Antibodies against JNK, p-ERK, ERK, p-p38, p-CaMKK, p-AMPK and p-IκB-α were bought from Cell Signaling Technology (Beverly, MA, USA). Antibodies that contrapose iNOS, NF-κB, IκBα, HO-1, Nrf2, p38 and β-actin were bought from Abcam (Cambridge, UK, USA). β-actin is used as an endogenous control protein.