Mouse anti nanog

Western blotting was carried out to detect the stem cell-related markers OCT3/4 and NANOG as well as MDR1, ATPase V₀a1 subunit, and TBP. Floating sphere or native cells were lysated with hot lysis buffer (1% SDS, Tris pH 7.4 20 mM, 5% β-mercaptoethanol) for the analysis of OCT3/4 and NANOG, or with RIPA buffer (Tris pH 7.6 50 mM, NaCl 150 mM, Triton-X 100 5%, sodium deoxycholate 0.25%, EGTA pH 8 1 mM, NaF 1 mM, Sigma) supplemented with protease inhibitors (Roche), for the other proteins. Equal amounts of protein lysates were subjected to reducing SDS-PAGE on a polyacrylamide gel, followed by to immunoblotting analysis. Blots were probed with a sheep anti-OCT3/4 (Abcam), a mouse anti-NANOG (Abcam), a mouse anti-MDR1 (D-11, Santa Cruz), a rabbit anti-ATPase V₀a1 (Abcam), or a rabbit anti-TBP (Santa Cruz) as reference. Incubation with horseradish peroxidase-conjugated secondary antibodies followed. The reaction was revealed by a chemiluminescence substrate (Pierce ECL Plus Western Blotting Substrate, Thermo Scientific). Immunoblot assays were repeated three times. The signal from each band was quantified by a dedicated software for densitometric evaluation (VisionWorksLS Analysis Software, Biospectrum, UVP).

The inserts of plasmids pcs2-UTX and pcs2-ZSCAN4D used in this study were PCR-amplified from the cDNA of NIH-3T3 cells and 2-cell mouse embryos, respectively. The PCR products were cloned into the Xho I and Xba I (Thermo, USA) sites downstream of the Myc ectopic tag. The antibodies covered were rabbit anti-UTX (Millipore, USA, for IF and WB), rabbit anti-H3K27me3 (Abcam, USA, for IF), mouse anti-Myc (Santa Cruz, USA, for IF), rabbit anti-ZSCAN4 (Millipore, for IF), rabbit anti-CDX2 (Abcam, for IF), mouse anti-NANOG (Abcam, for IF), rabbit α-tubulin (Proteintech, USA, for WB), Alexa Fluor 594 and Alexa Fluor 488 (Thermo, for IF), anti-UTX (KDM6A) (Abcam, for ChIP), anti-IgG (Abcam, for ChIP).