S., Lee H. DKC1 requires an additional coactivator activity (SCC-B) (promoter by OCT4 and SOX2 in vitro requires three distinct SCCs present in a human pluripotent cell nuclear extract (promoter template (Fig. 1B). These results demonstrated an important role of SCC-B in mediating cooperative coactivation by SCCs. Furthermore, our results suggested that the bulk of SCC-B likely resided in these fractions. Accordingly, SDSCpolyacrylamide gel electrophoresis (SDS-PAGE) of these Mono Emeramide (BDTH2) S fractions revealed that fractions 14 and 15 were highly enriched with a polypeptide at ~110 kDa along with multiple apparent breakdown products (Fig. 1C). Open in a separate window Fig. 1. Purification and identification of SCC-B as ATP-binding cassette subfamily F Emeramide (BDTH2) member 1 (ABCF1).(A) Chromatography scheme for purification of SCC-B from NT2 nuclear extracts (NT2 NE). NT2 NE is first subjected to ammonium sulfate precipitation (55% saturation) followed by a series of chromatographic columns including nickel affinity agarose (Ni-NTA), cation exchangers phosphocellulose (P11), heparin (Poros-HE), Mono S, anion exchanger Poros-HQ, hydroxyapatite (HAP), and gel filtration medium Superose 6. (B) Input fraction containing SCC-B activity from the Poros-HE step (IN), flow-through (FT), and various salt-eluted Mono S fractions was assayed for their ability to stimulate Mouse monoclonal to CD9.TB9a reacts with CD9 ( p24), a member of the tetraspan ( TM4SF ) family with 24 kDa MW, expressed on platelets and weakly on B-cells. It also expressed on eosinophils, basophils, endothelial and epithelial cells. CD9 antigen modulates cell adhesion, migration and platelet activation. GM1CD9 triggers platelet activation resulted in platelet aggregation, but it is blocked by anti-Fc receptor CD32. This clone is cross reactive with non-human primate OCT4/SOX2-dependent transcription from the human promoter template engineered with four extra copies of the oct-sox composite binding element (bottom). All reactions contain purified general transcription factors (GTFs), Pol II, OCT4, SOX2, and recombinant XPC and DKC1 complexes. Transcribed RNA products are subjected to primer extension and visualized by autoradiography. (C) Mono S fractions assayed in in vitro transcription are separated on a polyacrylamide gel and stained with silver. Filled arrowhead indicates the ~110-kDa polypeptide that comigrates with SCC-B transcriptional activity. (D) Coomassie staining of Mono S fraction 14 demonstrates purification to homogeneity. (E) ABCF1 is enriched in human ES cells. Down-regulation of Emeramide (BDTH2) ABCF1 in human ES cell line H9 upon exit from pluripotency. (F) ABCF1 is enriched in mouse ES cells. mRNA and protein levels of ABCF1 in pluripotent D3 mouse ES cells carrying V5 epitopeCtagged alleles (V5-ABCF1 knock-in) are compared to their differentiated counterparts. To identify these polypeptides that co-migrate with SCC-B activity, we pooled and separated peak Mono S fractions using SDS-PAGE. Tryptic digestion of the gel slice containing the 110-kDa protein band followed by mass spectrometry identified SCC-B to be ABCF1 (Fig. 1D). Identification of ABCF1 as the active constituent of SCC-B activity was unexpected because it has not been previously associated with transcriptional regulation or any cellular processes in the nucleus. To corroborate the mass spectrometry results, we showed by Western blotting that ABCF1 resides in nuclear and cytoplasmic fractions of human and mouse ES cells (fig. S1B), and is enriched exclusively in the phosphocellulose 1 M KCl (P1M) nuclear fraction that contains SCC activities, but not in the transcriptionally inactive P0.3 and P0.5 fractions (Fig. 1A and fig. S1C) (knockout mouse embryos die at 3.5 days post-coital, a developmental stage that coincides with the emergence of pluripotent cells in the inner cell mass of the blastocyst (gene loci in V5-ABCF1 knock-in D3 mouse ES cells. Representative data showing the enrichment of V5-ABCF1 (gray bars) compared to control IgGs (white bars) are analyzed by qPCR and expressed as percentage of input chromatin. Schematic diagrams of OCT4/SOX2 binding sites of each gene and the relative positions of the amplicons are shown at the bottom. (H) ABCF1.