ap2 alpha antibody (3b5)  (Bio-Techne corporation)

Journal: Cell reports

Article Title: Genetic and chromatin regulation of Pvt1 monoallelic expression

doi: 10.1016/j.celrep.2025.116554

Figure Lengend Snippet: (A) Allelic ratio (AR) of Pvt1 in F 1 -23 hybrid NPC clonal lines compared to other genes. Each point represents a clonal line ( n = 120). NPC clonal lines used for other experiments are highlighted with different shapes. (B) Summary of F 1 hybrid clonal lines used in this paper and their respective Pvt1 allelic expression status based on an AR cutoff of 0.2. (C) Allele-specific H2K27ac ChIP-seq with 3 different F 1 -23 clonal lines. Top row shows SNP differences between the 129 allele and the CAST allele in relation to the H3K27ac ChIP-seq signals across three samples. Tfap2a SNP is highlighted in the black box, the second of the two SNPs. (D) Allele-specific ATAC-seq and ChIP-seq from mE6 NPCs, clonal line with Pvt1 CAST monoallelic expression (mE6 NPC, blue). (E) Allele-specific ATAC-seq and ChIP-seq from Ch8 NPCs, clonal line with Pvt1 129 monoallelic expression (Ch8 NPC, pink). (F) ChIP input tracks (black) with allele-specific ChIP-seq tracks from mE6 NPCs. (G) ChIP input track (black) with allele-specific ChIP-seq tracks from Ch8 NPCs.

Article Snippet: Antibodies used: H3K27ac (Abcam, 4729), H3K27ac (Active Motif, 39133), H3K4me2 (Abcam, ab7766), H3K4me3 (Active Motif, 39159), H3K9me3 (Abcam, ab8898), H2AK199ub (Cell signaling, D27C4), H3K27me3 (Cell Signaling, C36B11), RNA Pol II-RPB1 (Bio-legend, 664906), and TFAP2a (Novus, NB100-74359).

Techniques: Expressing, ChIP-sequencing

Journal: Cell reports

Article Title: Genetic and chromatin regulation of Pvt1 monoallelic expression

doi: 10.1016/j.celrep.2025.116554

Figure Lengend Snippet: (A) Differential gene expression analysis (DESeq) between NPC clonal lines with Pvt1 CAST monoallelic expression ( n = 50) against NPC clonal lines with Pvt1 biallelic expression ( n = 58). Non-significant genes are in gray, and the significant genes based on log2 fold change and adjusted p value are in red, with the top 15 significant genes labeled. The adjusted p value cutoff is 0.01, and the log2 fold change cutoff is 0.5. (B) Tfap2a expression from F 1 -23 hybrid NPC clonal lines. Pvt1 allelic expression status is based on an AR cutoff of 0.2 ( n = 56, blue; n = 60, yellow; n = 4, red). Significance was calculated with a t test: NS p > 0.05 and ** p ≤ 0.01. (C) Summary of Tfap2a expression levels in transcript per million (TPM) between the different F 1 -23 clonal lines. The color of the clonal line is based on Pvt1 allelic status. (D) Tfap2a expression from RT-qPCR of R1-57 NPC clonal lines. Pvt1 allelic expression status is based on an AR cutoff of 0.2 ( n = 12 WT; n = 29, bi-allelic; n = 6, mutant). The relative fold change is compared to the NPC clonal line with the lowest Tfap2a expression. Significance was calculated with a t test: NS p > 0.05. (E) TFAP2a motif comparison between sequences from 129 allele (pink) and CAST allele (blue). (F) TFAP2a ChIP-seq tracks from four different samples: mE6 NPCs (blue), Ch8 NPCs (pink), Ch1 NPCs (yellow), and mESCs (green). The dotted line and black box around the SNPs demonstrate where the SNPs near the TFAP2a binding site can be found. (G) Violin plot of the AR from a non-clonal population of F 1 -23 NPCs ( n = 3) and a non-clonal population of F 1 -23 NPCs with Tfap2a overexpression ( n = 6, purple). Additionally, there is the AR of mE6 NPCs ( n = 3) and mE6 NPCs with Tfap2a overexpression ( n = 3, blue). The AR was obtained with targeted RNA-seq of Pvt1 . p values were calculated with an unpaired t test: *** p ≤ 0.001. The experimental schematic is provided in .

Article Snippet: Antibodies used: H3K27ac (Abcam, 4729), H3K27ac (Active Motif, 39133), H3K4me2 (Abcam, ab7766), H3K4me3 (Active Motif, 39159), H3K9me3 (Abcam, ab8898), H2AK199ub (Cell signaling, D27C4), H3K27me3 (Cell Signaling, C36B11), RNA Pol II-RPB1 (Bio-legend, 664906), and TFAP2a (Novus, NB100-74359).

Techniques: Gene Expression, Expressing, Labeling, Quantitative RT-PCR, Mutagenesis, Comparison, ChIP-sequencing, Binding Assay, Over Expression, RNA Sequencing

Journal: Cell

Article Title: DNA-guided transcription factor cooperativity shapes face and limb mesenchyme.

doi: 10.1016/j.cell.2023.12.032

Figure Lengend Snippet: Figure 3. Multiple homeodomains co-bind Coordinator motif with TWIST1 (A) Schematic of endogenous TF tagging and knockout. (B) Confirmation of TF tagging and depletion upon dTAGV-1 addition by western blot. IB, immunoblot. (C) Confirmation of ALX4 knockout in three independent clones by western blot. (D) Heatmap of TF binding (ChIP and CUT&RUN) and chromatin accessibility (ATAC) at promoter-distal binding sites for TWIST1 and/or AP-2a. Units: reads per genome coverage, except for ATAC, which is in signal per million reads. (E) The top enriched known motif for each TF, with p values. See also Figure S3.

Article Snippet: Antibodies used include TWIST1 (Abcam, ab50887), V5 (Abcam, ab15828), H3K27ac (Active Motif, 39133), Flag (Sigma-Aldrich, F1804), AP-2a (Cell Signaling, 3215), AP-2a (Novus Bio, NB10074359).

Techniques: Knock-Out, Western Blot, Clone Assay, Binding Assay

Journal: bioRxiv

Article Title: DNA-guided transcription factor cooperativity shapes face and limb mesenchyme

doi: 10.1101/2023.05.29.541540

Figure Lengend Snippet: A . Schematic of endogenous tagging TFs with the FKBP12 F36V degron and V5 epitope tag in human embryonic stem cells (hESC) followed by differentiation into cranial neural crest cells (hCNCC) and treatment with or without dTAG V -1. The ALX4 gene was instead knocked out by a frameshift mutation. B . Confirmation of TF tagging and depletion upon dTAG V -1 addition by Western blot for V5 epitope, with CTCF as a loading control. IB, immunoblot. C . Confirmation of ALX4 knockout in three independent clones by Western blot, with HSP90 as a loading control. D . Homeodomain TFs bind DNA at TWIST1-bound sites at variable occupancies. Heatmap shows promoter-distal binding sites for TWIST1 and/or AP2a. Assay indicates whether data shown is from ChIP, CUT&RUN (C&R), or ATAC, and whether an endogenous or V5 antibody was used. Rows are ranked by the sum of the homeodomain signals from undepleted cells. In the scale bar, units are reads per genome coverage, except for ATAC data, which is in signal per million reads. E . Homeodomain TFs bind the Coordinator motif. The top enriched motif (by AME) is shown for each TF, with p-values in parentheses.

Article Snippet: Antibodies used include TWIST1 (Abcam, ab50887), V5 (Abcam, ab15828), H3K27ac (Active Motif, 39133), Flag (Sigma-Aldrich, F1804), AP2a (Cell Signaling, 3215), AP2a (Novus Bio, NB100–74359).

Techniques: Mutagenesis, Western Blot, Control, Knock-Out, Clone Assay, Binding Assay