Anti nanog

Cells for immunocytochemistry were grown on ibidi µ-slides and stained as described in Kalmar et al. (2009 (link)). Primary antibodies were anti-NANOG (1:200; eBiosciences, 14-5761), anti-FLAG (1:1000; Sigma M2, F3165), anti-GATA6 (1:200; R&D, AF1700) and anti-GATA4 (1:200; Santa Cruz, sc-9053). Detection was performed using Alexa Fluor-conjugated secondary antibodies at 4 μg/ml (Molecular Probes). Nuclei were visualized using Hoechst 33342 dye at 100 μg/ml (Molecular Probes, H1399). Cells were imaged on a Zeiss LSM700 confocal microscope with a 40× oil immersion lens (NA 1.3).

Single-cell suspensions were rinsed twice and resuspended in phosphate-buffered saline (PBS) (10⁵ cells/100 μl) and subjected to FACS analysis as previously described [7 (link)]. In brief, for intracellular staining, cells were fixed in 70 % ethanol and subjected to 2 % human serum blocking of non-specific epitopes. Fluorescence-conjugated anti-CD44 (BD Biosciences, San Jose, CA, USA), anti-SOX2 and anti-BMI1 (R&D Systems, Minneapolis, MN, USA), and anti-OCT4 and anti-NANOG (ebiosciences, San Diego, CA, USA) antibodies were used in this study. Respective IgG isotypic controls were included in the experiment. At least 10,000 cells were acquired for each test sample and analyzed with a BD FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and Flowjo software (Treestar/ Flowjo, LLC, Ashland, OR, USA).

Cells were washed twice with phosphate-buffered saline (PBS, Nacalai Tesque, 14249-24), trypsinized, and collected by centrifugation at 190 × g for 2 min at 20 °C. The cells were counted and washed twice with PBS. The cells were then lysed in lysis buffer (0.5% Triton X-100 (Sigma-Aldrich, T8787-100ML), 150 mM NaCl (Wako, 191-01665), and 20 mM Tris-HCl [pH 7.5]) to obtain 2 × 10⁶ cells per 100 µL. The lysates were then incubated at 95 °C for 5 min and filtered using a QIAshredder homogenizer (Qiagen, 79656). The extracted proteins were analyzed using 5–20% gradient sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto Immobilon Transfer Membranes (Millipore, INYC00010) for immunoblotting. The primary antibodies used were anti-mNeonGreen (1:500; Chrom Tech, Apple Valley, MN, USA, 32f6-100, RRID:AB_2827566), anti-GAPDH (1:5000; Cell Signaling Technology, Danvers, MA, USA, 5174, RRID:AB_10622025), anti-NANOG (1:1000; eBioscience, San Diego, CA, USA, 14-5761-80, RRID:AB_763613), anti-SOX2 (1:1000; Abcam, Cambridge, UK, ab97959, RRID:AB_2341193), and anti-USP5 (1:2000; 10473-1-AP, Proteintech, Rosemont, IL, USA, RRID:AB_2272754).

Western blot was performed as we previously reported [25 (link)]. The antibodies used in this article are listed as follows: anti-p57 (1 : 500, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-p53 (1 : 500, Wanleibio, Xi'an, China), anti-p-p53 (1 : 500, Wanleibio, Shenyang, China), anti-GAPDH (1 : 5000; Genesci, Beijing, China), anti-PCNA (1 : 1000, Boster, Wuhan, China), anti-Cyclin A (1 : 300, Santa Cruz, Dallas, Texas, USA), anti-Cyclin E (1 : 300, Santa Cruz), anti-OCT4 (1 : 500, Santa Cruz), anti-NANOG (1 : 500, PeproTech, Rocky Hill, New Jersey, USA), anti-SOX2 (1 : 1000, Proteintech Group, Rosemont, Illinois, USA), horse-radish peroxidase-conjugated anti-rabbit antibody (1 : 3000, Boster), and anti-mouse antibody (1 : 2000; Boster).