Anti sirt1

Cell lysates and tissue samples containing equal amounts of protein underwent SDS-PAGE gel, transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), and immunoblotted with a primary antibody, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibodies. HRP substrate peroxide solution/luminol reagents (Immobilon Western Chemiluminescent Substrate, Millipore) were added and the secondary antibody signals on membranes were visualized with the Fujifilm LAS4000 luminescent image analysis system. The following primary antibodies were used: anti-p53, anti-p21, anti-p16, anti-SIRT1, anti-caspase 3, anti-PARP, anti-A_2AR, anti-Tuj1, anti-iNOS, anti-calretinin, and anti-NeuN (GeneTex); anti-phospho-Rb, anti-phospho-Akt, anti-Rb, and anti-Akt (Cell Signaling Technology); anti-doublecortin (DCX) and anti-ionized calcium binding adapter molecule 1 (Iba1) (Abcam); and anti-glial fibrillary acidic protein (GFAP) (Millipore). All antibodies were used at dilution 1:1,000.

Cisplatin, amifostine (AMF), adenosine, and N6-(2′-hydroxyethyl) adenosine (HEA), as well as other reagents and solvents, were obtained from Sigma-Aldrich (St. Louis, MO, USA). Commercial assay kits for BUN and CRE were provided by HUMAN Diagnostics Worldwide (Wiesbaden, Germany). Mouse TNF-α, IL-1β, and IL-6 ELISA Max™ Set Deluxe Kits were purchased from BioLegend Inc. (San Diego, CA, USA). Primary antibodies for Western blots against anti-COX-2, anti-p-JNK, anti-catalase, anti-GPx, anti-SOD, anti-AMPK, anti-Sirt1, anti-TLR4, anti-P62, anti-Beclin-1, anti-cleaved PARP, and anti-cleaved caspase were purchased from GeneTex (San Antonio, TX, USA). Anti-OAT1 and anti-OAT3 were purchased from ABclonal (MA, USA). Antibodies against anti-JNK, anti-p-ERK, anti-ERK, anti-p-p38, anti-p-IκBα, and anti-LC3B were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against anti-iNOS, anti-NF-κB, anti-IκBα, anti-p38, anti-HO-1, anti-Nrf-2, and β-actin were purchased from Abcam (Cambridge, UK). Protein assay kits (Bio-Rad Laboratories Ltd., Watford, Herts, UK) were obtained as indicated.