Irdye 680 goat anti mouse or anti rabbit igg

AN446 was synthesized as described [10 ]; HDAC inhibitors—SAHA and entinostat (MS-275)—were obtained from Cayman Chemical (Ann Arbor, MI, USA), romidepsin (depsipeptide) from ApexBio Tech LLC (Houston, TX, USA), and panobinostat (LBH589) and belinostat (PXD101) from MedChem Express (Princeton, NJ, USA). These HDACIs were dissolved in DMSO to a concentration of 100 mM and stored in aliquots at −20 °C. Dox hydrochloride 2 mg/mL, was obtained from Ebewe Pharma Ges.m.b.H. (Unterach, Austria) and was diluted in saline solution. Polyclonal antibodies: c-Myc #9402, SIRT1 #2310 (Cell Signaling, Danvers, MA, USA) and superoxide dismutase 1(SOD1)#ab13498 (Abcam, Cambridge, UK), phospho-H2AX, Serine 139, pH2AX # A300-081A-M (Bethyl Laboratories, Montgomery, TX, USA), cyclooxygenase-2 (COX-2) #PA5-16817 (ThermoFisher, Rockford, IL, USA), Ki-67, BRB040 (Zytomed, Berlin, Germany). Mouse monoclonal antibodies: Rad51 #05-530-I (Sigma, Saint Louis, MO, USA), and actin # SKU:0869100-CF (MP Biomedicals, Aurora, OH, USA). Secondary antibodies: IRDye^® 680 goat anti-mouse or anti-rabbit IgG (LI-COR Biosciences, Lincoln, NE, USA), and biotinylated goat anti-rabbit IgG-B (Santa Cruz Biotechnology, CA, USA).

Real-time RT-PCR and western blot were performed as previously reported 12 (link). Briefly, total RNA was extracted and according to the manufacturer's instructions, cDNA synthesis was performed using a PrimeScript™ RT reagent kit and real-time PCR was performed using a SYBR® Premix Ex Taq™ kit (Takara, Dalian, China). Primer sequences are shown in Supplementary Table S1. As for western blot, mouse anti-eIF4A3, mouse anti-STAU1, mouse anti-TPI1, mouse anti-PRDX6, mouse anti-human β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-HA or Flag (Proteintech, Wuhan, China) were used as the primary antibodies and IRDye 680 goat anti-mouse or anti-rabbit IgG were the secondary antibody (LI-COR Biosciences, Lincoln, NE, USA). Protein relative levels were measured with the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).