anti tfap2c  (Proteintech)

Journal: Science Advances

Article Title: Specialized signaling centers direct cell fate and spatial organization in a mesodermal organoid model

doi: 10.1126/sciadv.ady7682

Figure Lengend Snippet: ( A ) Schematic describing embryonic stages of mouse limb bud formation and the main cell populations involved in limb morphogenesis. ( B ) Schematic of the protocol generating cells with AER, surface ectoderm, and mesoderm properties from mESCs. ( C ) Representative confocal image of generated AER-like cells in day 7 cultures. Cyan: Hoechst; white: phalloidin; magenta: TP63; yellow: Fgf8:tdTomato . Scale bar, 100 μm. ( D ) Representative confocal image of day 7 heterogeneous cultures stained for immature surface ectoderm TFAP2C and basal epithelial stem cell marker TP63. Cyan: Hoechst; magenta: TP63; white: TFAP2C. Scale bar, 400 μm. ( E ) Bar plot showing flow cytometry quantification of epithelial cell marker EpCAM+ cells in day 7 cultures. Each dot represents one biological replicate. N = 9. ( F ) Bar plot showing flow cytometry quantification of Fgf8:tdTomato + cells in day 7 cultures. Each dot represents one biological replicate. N = 4. ( G ) UMAP representation of scRNA-seq data of day 7 cultures displaying main cell clusters. See (H) color-coding. ( H and I ) Plots of the scRNA-seq cluster–based proportions of (H) generated cell types, with color-coding for AER-like, mesoderm-like, and surface ectoderm cells, and (I) lineages on day 7. For full information, see fig. S2E. Rep denotes replicate. ( J ) Dot plot showing surface ectoderm, AER, and limb bud mesoderm marker gene expression in the scRNA-seq clusters. Dot size represents the percentage of cells expressing each marker. ( K ) Representative confocal image of day 7 heterogeneous cultures generated from the Prrx1 Enh :mVenus reporter mESC line. White: phalloidin; magenta: TP63; green: Prrx1 Enh :mVenus . Scale bar, 500 μm. ( L ) Representative sequential z -stack confocal images of domes in day 7 cultures. Magenta: TP63; green: Prrx1 Enh :mVenus . Images from left to right show the dome from bottom to top, denoted by z numbers. Scale bars, 200 μm.

Article Snippet: The samples were then incubated with primary antibodies [TP63 (Abcam, ab735), TFAP2C (Cell Signaling, 2320S), LEF1 (Abcam, ab137872)], diluted in blocking buffer overnight at 4°C.

Techniques: Generated, Staining, Marker, Flow Cytometry, Gene Expression, Expressing

Journal: PLOS Pathogens

Article Title: A genome-wide analysis of YY1 and TFAP2 competition on overlapping motifs reveals their roles in HPV-induced carcinogenesis

doi: 10.1371/journal.ppat.1013524

Figure Lengend Snippet: (A-C) Identification and validation of proteins binding to a YY1-binding motif B. The wild-type YY1 motif containing oligo 4 (oligo 4) and its mutant oligo 4 (oligo 4-M) were used for the pulldown assays with the nuclear extracts from CaSki cells with or without siRNA knockdown of HPV16 E7 expression (siE7, E7-specific siRNA; siNS, nonspecific siRNA control). (A) Workflow of DNA oligo pulldown and mass-spectrometry assays. (B) Knockdown (KD) of E7 in CaSki cells was performed as described previously . The soluble nuclear extract (Nuc in red) was isolated by fractionation and used for DNA oligo pulldown assays in panels C-E. The KD efficiency of HPV16 E7 was indicated by stabilized and increased p53 protein level. Fractionation efficiency was evaluated by immunoblot of tubulin in the cytoplasm (Cyto) and hnRNP C1/C2 in the nucleus. (C) A scatter plot shows the peptide counts for proteins on oligo4 over that on oligo4-M which were identified by mass spectrometry. (D-F) TFAP2 binds to the YY1 motif B, but not the motif A. (D) Binding of TFAP2A and TFAP2C to the YY1 motif B. The oligo4 with YY1 motif B and oligo4-M with a mutant motif B were used for the pulldown and Western blot analyses using fractionated nuclear extracts isolated from the CaSki cells with indicated siRNA KD. (E) YY1 motif A in lnc-FANCI-2 promoter binds YY1, but not TFAP2A and TFAP2C. Pulldown assays of oligo5 containing YY1 motif A and its mutant oligo5-M were performed as described for oliog4 pulldown and Western blot analyses. (F) YY1 motif B binds to both TFAP2A and TFAP2C independent of YY1. The YY1-motif B containing Oligo 4 was used for the pulldown assays with soluble nuclear extracts fractionated from CaSki cells treated with non-specific siRNA (siNS), YY1 siRNA (siYY1) or E7 siRNA (siE7). KD efficiency was measured by stabilized p53 protein.

Article Snippet: Anti-YY1 (#66281–1-Ig), anti-TFAP2C (#14572–1-AP), and anti-p53 (#10442–1-AP) antibodies were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Biomarker Discovery, Binding Assay, Mutagenesis, Knockdown, Expressing, Control, Mass Spectrometry, Isolation, Fractionation, Western Blot