Anti klf4

For flow cytometry, the cells were treated with the FIX & PERM^® Kit (Thermo Fisher Scientific, MA, USA) and then, incubated for 1 h at RT with anti-Oct-4 Alexa Fluor 488 conjugated (Cell Signaling, Danvers, MA, USA) 1:50, anti-Nanog Alexa Fluor 647 conjugated (Becton Dickinson, Franklin Lakes, NJ, USA) 1:20, anti-c-Kit PE conjugated (Becton Dickinson, NJ, USA) 1:5, anti-Sox2 Alexa Fluor 488 conjugated (Becton Dickinson, NJ, USA) 1:5, anti-OVOL-1 (Abcam, Cambridge, UK) 1 µg/mL, anti-KLF4 (Thermo Fisher Scientific, Waltham, MA, USA) 1:1000, anti-ESG1 (Abcam, Cambridge, UK) 1:50, anti-REX1 (Abcam, Cambridge, UK) 1:200 or appropriate isotype controls (all from Becton Dickinson, NJ, USA), followed by appropriate secondary antibody Alexa Fluor 488 if necessary (1:100, Invitrogen, Carlsbad, CA, USA). Cytometric analyses were performed with a Cytoflex cytometer (Beckman Coulter, CA, USA), and the data were analyzed with CytExpert Acquisition and Analysis Software (Beckman Coulter, CA, USA).

60 μg of total protein was separated on a 10% SDS-PAGE gel according to standard procedure, and the transfer was performed on a 0.45 μm nitrocellulose membrane (Thermo Scientific); the membrane was blocked with 5% low-fat milk for 1 h. Subsequently, the membranes were incubated with the different antibodies: anti-OCT4 (Santa Cruz Biotechnology, sc-5279), anti-SOX2 (Santa Cruz Biotechnology, sc-365823), anti-KLF4 (Thermo Fisher Scientific, MA5-15672), anti-C-MYC (Santa Cruz Biotechnology, sc-40), and anti-NANOG (Santa Cruz Biotechnology, sc-376915). Then, the membranes were incubated with an anti-mouse secondary antibody (Santa Cruz Biotechnology, sc-2005). As a loading control, β-ACTIN (Santa Cruz Biotechnology, sc-47778) was used. For detection of the protein-antibody complex, we used the Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500) according to the manufacturer's specifications and visualized it with C-DiGit equipment (Li-Cor). Data were presented as relative expression protein levels normalized for β-ACTIN protein.