anti pan lap2  (MedChemExpress)

Journal: Science Advances

Article Title: DLX2 acts as a pioneer factor and drives Msx1 + ectomesenchyme formation from embryonic stem cells

doi: 10.1126/sciadv.aea0685

Figure Lengend Snippet: ( A ) Schematic of FLAG-tagged DLX2-binding partner identification and validation. m/z , mass/charge ratio; WB, Western blot. ( B ) Liquid chromatography–mass spectrometry (LC-MS) heatmap of day-2 Dlx2 -OE (FLAG +Dox) EBs. Proteins were labeled by UniProt entry names, with FC indicated by color intensity (red). ( C ) Immunostaining of Dlx2 -OE cells. DAPI for nuclear staining. Scale bars, 50 μm (EBs) and 10 μm (single cells). ( D and E ) Co-IP of DLX2 and LAP2 isoforms in day-2 Dlx2 -OE EBs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. ( F ) Schematic of DLX2 truncation mutants: WT and four mutants with deletions in the N-terminal (ΔN), C-terminal (ΔC), homeodomain (ΔHD), or 38 amino acids in the homeodomain, retaining the NLS. aa, amino acids. ( G ) Co-IP of LAP2α, LAP2β, and FLAG-DLX2 mutants in day-2 EBs using anti-FLAG antibody. ( H ) Predicted DLX2-LAP2α protein complex structure. ( I ) Flow cytometry quantification of Msx1 + cells in day-8 EBs. Data shown as mean ± SD from three independent experiments. Statistics: one-way ANOVA with Dunnett’s post hoc test. ( J ) RT-qPCR of ectomesenchymal marker genes in day-8 EBs. Data shown as mean ± SD from three independent experiments. Statistics: one-way ANOVA with Dunnett’s post hoc test. ( K ) RT-qPCR of Tmpo KD efficiency in day-8 Dlx2 -OE EBs, with two shRNA mix delivered into cells. Data shown as mean ± SD from three independent experiments. Statistics: two-tailed unpaired Student’s t test. ( L ) Flow cytometry quantification of Msx1 + cells in day-8 Dlx2 -OE EBs with Tmpo KD. Data shown as mean ± SD from four independent experiments. Statistics: one-way ANOVA with Dunnett’s post hoc test. ( M ) RT-qPCR of ectomesenchymal marker genes in day-8 Dlx2 -OE EBs with Tmpo KD. Data shown as mean ± SD from three independent experiments. Statistics: two-tailed unpaired Student’s t test. ** P < 0.01, *** P < 0.001, and **** P < 0.0001. n.s., not significant.

Article Snippet: After preclearing with magnetic beads, the lysates were incubated overnight at 4°C with anti-FLAG, anti-pan LAP2, anti-H3K27ac, or control IgG antibodies, followed by a 4-hour incubation with Protein G or Protein A/G magnetic beads (Invitrogen, catalog no. 10004D; MedChemExpress, catalog no. HY-K0202).

Techniques: Binding Assay, Biomarker Discovery, Western Blot, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, Immunostaining, Staining, Co-Immunoprecipitation Assay, Control, Flow Cytometry, Quantitative RT-PCR, Marker, shRNA, Two Tailed Test

Journal: Science Advances

Article Title: DLX2 acts as a pioneer factor and drives Msx1 + ectomesenchyme formation from embryonic stem cells

doi: 10.1126/sciadv.aea0685

Figure Lengend Snippet: ( A ) Schematic of integrative analysis combining RNA-seq and CUT&Tag data. ( B ) DEG analysis between Dlx2 - and GFP-OE control cells. DEGs were identified with DESeq2 (log 2 FC ≥ 2 and P < 0.05) and included multiple ectomesenchymal markers. ( C ) GO analysis of up-regulated genes in Dlx2 -OE cells. The top 20 GO terms displayed in the dot plot. ( D ) Heatmap of a genome-wide overview of DLX2-binding sites identified by CUT&Tag. Each row represented a 3-kb window centered on the peak midpoint. ( E ) Pie chart showing the genomic distribution of DLX2-binding sites. 3′UTR, 3′ untranslated region. ( F ) HOMER analysis of known DNA sequence motifs enriched at significant DLX2-binding peaks. ( G ) Venn diagram showing overlap of promoter-proximal DLX2- and LAP2α-binding targets with DEGs from RNA-seq. ( H ) Representative Integrative Genomics Viewer tracks of DLX2 and LAP2α signals at promoter regions of target genes. ( I ) KD efficiency of target genes assessed by RT-qPCR in day-8 Dlx2 -OE EBs, with three shRNA mix delivered via the PB transposon system. Error bars represented data as mean ± SD from three independent experiments. Statistics: two-tailed unpaired Student’s t test. ( J and K ) Flow cytometry analysis of Msx1 + cell proportions in day-8 EBs after KD of target genes, showing representative plots (J) and quantitative FC in Msx1 low and Msx1 high populations relative to scramble control (K). Error bars represented data as mean ± SD from three independent experiments. Statistics: one-way ANOVA with Dunnett’s post hoc test. ( L ) RT-qPCR of ectomesenchymal marker gene expression in day-8 Dlx2 -OE EBs with Dlx6 , Hmga2 , Msx2 , or Gata4 KD. Heatmap showed relative expression levels compared to scramble control: blue (<1), and pink (>1). Data represented mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. n.s., not significant.

Article Snippet: After preclearing with magnetic beads, the lysates were incubated overnight at 4°C with anti-FLAG, anti-pan LAP2, anti-H3K27ac, or control IgG antibodies, followed by a 4-hour incubation with Protein G or Protein A/G magnetic beads (Invitrogen, catalog no. 10004D; MedChemExpress, catalog no. HY-K0202).

Techniques: RNA Sequencing, Control, Genome Wide, Binding Assay, Sequencing, Quantitative RT-PCR, shRNA, Two Tailed Test, Flow Cytometry, Marker, Gene Expression, Expressing

Journal: Science Advances

Article Title: DLX2 acts as a pioneer factor and drives Msx1 + ectomesenchyme formation from embryonic stem cells

doi: 10.1126/sciadv.aea0685

Figure Lengend Snippet: Through the formation of a DLX2-LAP2α-nucleosome complex that remodeled chromatin, DLX2 drove ectomesenchymal specification in mESCs, generating Msx1 + progenitors with craniofacial regeneration potential.

Article Snippet: After preclearing with magnetic beads, the lysates were incubated overnight at 4°C with anti-FLAG, anti-pan LAP2, anti-H3K27ac, or control IgG antibodies, followed by a 4-hour incubation with Protein G or Protein A/G magnetic beads (Invitrogen, catalog no. 10004D; MedChemExpress, catalog no. HY-K0202).

Techniques: