Rabbit anti nanog

For the detection of protein expression in ESCs and EBs, samples were lysed in RIPA buffer supplied with 1 mM PMSF (Li & Wang, 2011 (link)). 40–80 μg of cell lysates was processed with SDS–PAGE. The following primary antibodies were used: rabbit anti-Upf1 (1:1,000, Bethyl Laboratories, Montgomery, TX, USA), rabbit anti-Upf2 (1:1,000, New England Biolabs), rabbit anti-Oct4 (1:1,000; New England Biolabs), mouse anti-Oct4 (1:6,000; Santa Cruz, Heidelberg, Germany), rabbit anti-Nanog (1:5,000; Merck-Millipore), rabbit anti-Smg6/Est1A (1:1,500; Abcam, Cambridge, UK), rabbit anti-p-Smad2/3 (1:1,000; New England Biolabs), rabbit anti-β-Catenin (1:800; Sigma-Aldrich), mouse anti-β-Tubulin (1:2,000; Sigma-Aldrich), and mouse anti-Actin (1:10,000; Sigma-Aldrich). The secondary antibodies used in these studies were HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:2,000; DAKO, Hamburg, Germany). ECL Western blotting substrates (Thermo Scientific, Rockford, IL, USA) were used for HRP detection.

The StemCCA lentiviral vector under a constitutive EF1α promoter is used to reprogram MEFs to iPSCs (Sommer et al, 2009 (link)). Briefly, lentiviruses were produced by transfecting 293T cells with three plasmids—pMD2.G, psPAX2, and StemCCA lentiviral vector. Viruses were collected 36 h post-transfection and applied onto 1 × 10⁵ MEFs in 6-well plates. To determine the reprograming efficiency, the iPSC cultures were stained with the AP staining kit on the indicated days. Single clones were selected for expansion and then further characterized for the expression of stem cell markers by Western blotting. The following antibodies were used: rabbit anti-Oct4 (1:1,000; Cell Signaling), rabbit anti-Sox2 (1:1,000; Abcam), rabbit anti-Nanog (1:1,000; Merck-Millipore), and anti-β-Actin (1:10,000; Sigma-Aldrich). For teratoma induction, each iPSC line was inoculated subcutaneously four points (1 × 10⁶ cells/point) in 4- to 5-week-old male immune-deficient CD1 nude mice. Eighteen days later, tumors were fixed overnight in 4% PFA, sectioned, and stained with H&E to determine derivatives of different germ layers based on the morphological characteristics of cells in tumors/teratomas. iPSC clones were injected into NMR1 blastocysts to generate chimeric mice.