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mouse anti-aff4 antibody (Abnova)

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Abnova mouse anti-aff4 antibody
Mouse Anti Aff4 Antibody, supplied by Abnova, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse anti-aff4 antibody/product/Abnova
Average 90 stars, based on 1 article reviews

mouse anti-aff4 antibody - by Bioz Stars, 2026-05

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Figure 1. Functional Epigenome-wide Screen Identiﬁes AFF4 as an Epigenetic Dependency in H3K27M+ DMG (A) Pooled epigenomic shRNA library is transduced into SU-DIPG4 cells, and genomic DNA is isolated and sequenced to identify individual shRNA ampliﬁcation or loss. (B) Aggregate normalized sequencing data showing depleted shRNAs (fold change % 0.8 in red). (C) Leading edge of depleted shRNAs ordered by fold change. (D) Secondary validation of screening hits by growth rate (SF8628, left) or colony formation (SF7761, right). Data are displayed as mean ± SD. (E) Model of the SEC in which CDK9 phosphorylates the C-terminal domain of RNA Pol II, resulting in release from promotor-proximal pause site. Experiments in (A)–(C) reﬂect n = 3 independent biological replicates. Experiments in (D) were performed with minimum n = 3 technical replicates.

Journal: Cell reports

Article Title: Super Elongation Complex as a Targetable Dependency in Diffuse Midline Glioma.

doi: 10.1016/j.celrep.2020.03.049

Figure Lengend Snippet: Figure 1. Functional Epigenome-wide Screen Identiﬁes AFF4 as an Epigenetic Dependency in H3K27M+ DMG (A) Pooled epigenomic shRNA library is transduced into SU-DIPG4 cells, and genomic DNA is isolated and sequenced to identify individual shRNA ampliﬁcation or loss. (B) Aggregate normalized sequencing data showing depleted shRNAs (fold change % 0.8 in red). (C) Leading edge of depleted shRNAs ordered by fold change. (D) Secondary validation of screening hits by growth rate (SF8628, left) or colony formation (SF7761, right). Data are displayed as mean ± SD. (E) Model of the SEC in which CDK9 phosphorylates the C-terminal domain of RNA Pol II, resulting in release from promotor-proximal pause site. Experiments in (A)–(C) reﬂect n = 3 independent biological replicates. Experiments in (D) were performed with minimum n = 3 technical replicates.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies AFF4 mouse mAb (Western) Santa Cruz Santa Cruz (G-1): sc-390310 AFF4 rabbit pAb (Co-IP) Atlas Antibodies HPA029634; RRID: AB_2673129 CDK9 rabbit mAb Abcam Abcam [EPR3119Y] (ab76320); RRID:AB_1267054 phospho-Rbp1 CTD (Ser2) rabbit mAb Cell Signaling Cell Signaling (E1Z3G) #13499 phospho-Rbp1 CTD (Ser5) rabbit mAb Cell Signaling Cell Signaling (D9N5I) #13523S a-Tubulin mouse mAb Cell Signaling Cell Signaling (DM1A) #3873S Olig2 rabbit Ab Immuno-Biological IBL #18953; RRID:AB_2267671 Histone 3.3, K27M mutant rabbit pAb Millipore EMD Millipore ABE419; RRID:AB_2728728 Histone H3 rabbit pAb Active Motif Active Motif #39163; RRID:AB_2614978 Histone H3 (acetyl K27) rabbit pAb (ChIP grade) Abcam Abcam (ab4729) Tri-Methyl-Histone H3 (Lys27) rabbit mAb Cell Signaling Cell Signaling (C36B11) #9733 RNA polymerase II CTD mouse mAb (ChIP grade) Abcam Abcam [8WG16] (ab817); RRID:AB_306327 Chemicals, Peptides, and Recombinant Proteins Atuveciclib (BAY-1143572) MedChemExpress HY-12871; CAS 1414943-88-6 AZD4573 MedChemExpress HY-112088; CAS 2057509-72-3 PHA-767491 SelleckChem S2742 CAS 942425-68-5 Flavopiridol SelleckChem S1230 CAS 146426-40-6 LDC000067 SelleckChem S7461 CAS 1073485-20-7 Critical Commercial Assays RNeasy Plus Mini Kit QIAGEN Cat #74136 Aldefluor Kit Stemcell Cat #01700 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat #4368813 Deposited Data ChIP sequencing mapped raw data This paper GEO: GSE129777 RNA sequencing mapped raw data This paper GEO: GSE129777 Neuroepithelial stem cell ChIP-seq reference data ENCODE project https://www.encodeproject. org/reference-epigenomes/ ENCSR978RSW/ ENCODE reference epigenome brain ChIP-seq ENCODE project https://www.encodeproject. org/search/?type= ReferenceEpigenome Published SU-DIPG4 and SF8628 ChIP-seq data Piunti et al., 2017 GEO: GSE78801 Published iOPC and DIPG patient ChIP-seq data Nagaraja et al., 2019 GEO: GSE126319 Experimental Models: Cell Lines SU-DIPG4 Michelle Monje (Nagaraja et al., 2017) RRID:CVCL_IP14 SU-DIPG13 Michelle Monje (Nagaraja et al., 2017) N/A HSJD-DIPG007 Angel Montero Carcaboso N/A SF7761 Nalin Gupta (Olow et al., 2016) RRID:CVCL_IT45 SF8628 Nalin Gupta (Olow et al., 2016) RRID:CVCL_IT46 BT245 Adam Green (Green et al., 2015) RRID:CVCL_IP13 (Continued on next page) e1 Cell Reports 31, 107485, April 7, 2020

Techniques: Functional Assay, shRNA, Isolation, Sequencing, Biomarker Discovery

Figure 2. SEC Is Critical for DMG Cell Proliferation and Clonogenic Potential (A) Decrease in AFF4 mRNA levels in SU-DIPG4, HSJD-DIPG007, BT245, and SF8628 cell lines following transduction with two non-overlapping shRNA lentiviral constructs in comparison to shNull control (two-tailed t test; *p < 0.05; **p < 0.001; ***p < 0.0001). (B) Clonogenic potential following shAFF4 knockdown as measured by adherent colony formation (two-tailed t test; *p < 0.05; **p < 0.001; ***p < 0.0001). (C) Rate of adherent growth in SU-DIPG4, HSJD-DIPG007, and SF8628 cells treated with shNull or shAFF4. Data are displayed as mean ± SEM. (D) Representative ﬂuorescent live-cell imaging from HSJD-DIPG007 cells following respective lentiviral transduction (scale bars, 400 mm). (E) Co-immunoprecipitation with either anti-AFF4 or immunoglobulin G (IgG) control antibodies followed by immunoblot for P-TEFb member proteins. (F) Rate of growth in BT245, HSJD-DIPG007, and SF8628 cells treated with shNull or shCDK9 constructs. Data are displayed as mean ± SEM. Experiments in (A)–(D) and (F) were repeated for at least two cell lines, with all data shown representing minimum n = 3 replicates.

Journal: Cell reports

Article Title: Super Elongation Complex as a Targetable Dependency in Diffuse Midline Glioma.

doi: 10.1016/j.celrep.2020.03.049

Figure Lengend Snippet: Figure 2. SEC Is Critical for DMG Cell Proliferation and Clonogenic Potential (A) Decrease in AFF4 mRNA levels in SU-DIPG4, HSJD-DIPG007, BT245, and SF8628 cell lines following transduction with two non-overlapping shRNA lentiviral constructs in comparison to shNull control (two-tailed t test; *p < 0.05; **p < 0.001; ***p < 0.0001). (B) Clonogenic potential following shAFF4 knockdown as measured by adherent colony formation (two-tailed t test; *p < 0.05; **p < 0.001; ***p < 0.0001). (C) Rate of adherent growth in SU-DIPG4, HSJD-DIPG007, and SF8628 cells treated with shNull or shAFF4. Data are displayed as mean ± SEM. (D) Representative ﬂuorescent live-cell imaging from HSJD-DIPG007 cells following respective lentiviral transduction (scale bars, 400 mm). (E) Co-immunoprecipitation with either anti-AFF4 or immunoglobulin G (IgG) control antibodies followed by immunoblot for P-TEFb member proteins. (F) Rate of growth in BT245, HSJD-DIPG007, and SF8628 cells treated with shNull or shCDK9 constructs. Data are displayed as mean ± SEM. Experiments in (A)–(D) and (F) were repeated for at least two cell lines, with all data shown representing minimum n = 3 replicates.

Techniques: Transduction, shRNA, Construct, Comparison, Control, Two Tailed Test, Knockdown, Live Cell Imaging, Immunoprecipitation, Western Blot

Figure 3. AFF4 Depletion Induces Transcriptional Proﬁles of Cellular Differentiation and Decreases Potential for Self-Renewal (A) Unsupervised hierarchical clustering of 1,671 differentially expressed genes from BT245 cells treated with either shNull or shAFF4 (n = 3; fold change </> 2 and p < 0.01). (B and C) GSEA results (B) and select enrichment plots (C) of differentiation- and Myc-associated gene sets, ordered by normalized enrichment score (NES). (D) Differentially expressed genes in differentiation-, stemness-, or Myc target-associated gene sets as deﬁned by GSEA. Select illustrative genes with fold change </> 1 and p < 0.01 are labeled. (E) Neurosphere formation efﬁcacy by extreme limiting dilution assay (c2 shNull versus shAFF4 no. 1 p = 0.035 versus shAFF4 no. 2 p = 0.008). (F) Normalized growth in neurosphere size of cells grown in suspension following transduction with shRNA constructs. Data are displayed as mean ± SEM (two- tailed t test; *p < 0.05; **p < 0.001). (G) Representative images of neurospheres from SU-DIPG4 cells transduced with shRNA constructs containing a GFP reporter (scale bars, 400 mm). Experiments in (A)–(E) were performed with n = 3 technical replicates. Experiments in (F) and (G) were independently repeated for at least two cell lines with minimum n = 3 technical replicates.

Journal: Cell reports

Article Title: Super Elongation Complex as a Targetable Dependency in Diffuse Midline Glioma.

doi: 10.1016/j.celrep.2020.03.049

Figure Lengend Snippet: Figure 3. AFF4 Depletion Induces Transcriptional Proﬁles of Cellular Differentiation and Decreases Potential for Self-Renewal (A) Unsupervised hierarchical clustering of 1,671 differentially expressed genes from BT245 cells treated with either shNull or shAFF4 (n = 3; fold change 2 and p < 0.01). (B and C) GSEA results (B) and select enrichment plots (C) of differentiation- and Myc-associated gene sets, ordered by normalized enrichment score (NES). (D) Differentially expressed genes in differentiation-, stemness-, or Myc target-associated gene sets as deﬁned by GSEA. Select illustrative genes with fold change 1 and p < 0.01 are labeled. (E) Neurosphere formation efﬁcacy by extreme limiting dilution assay (c2 shNull versus shAFF4 no. 1 p = 0.035 versus shAFF4 no. 2 p = 0.008). (F) Normalized growth in neurosphere size of cells grown in suspension following transduction with shRNA constructs. Data are displayed as mean ± SEM (two- tailed t test; *p < 0.05; **p < 0.001). (G) Representative images of neurospheres from SU-DIPG4 cells transduced with shRNA constructs containing a GFP reporter (scale bars, 400 mm). Experiments in (A)–(E) were performed with n = 3 technical replicates. Experiments in (F) and (G) were independently repeated for at least two cell lines with minimum n = 3 technical replicates.

Techniques: Cell Differentiation, Labeling, Limiting Dilution Assay, Suspension, Transduction, shRNA, Construct, Two Tailed Test

Figure 5. The H3K27M Mutation Alters Epigenetic Regulation of AFF4 Promoter (A) H3K27ac deposition at the AFF4 promoter in neuroepithelial stem cells, OPCs, and differentiated brain regions (identical reads and scale for each sample tract). (B) H3K27ac deposition at the AFF4 promoter in patient-derived DMG cultures (identical reads and scale for each sample tract). (C) AFF4 mRNA transcripts (left) and protein expression (right) in H3F3A wild-type HSJD-GBM01 and normal human astrocytes following lentiviral transduction of the H3K27M transgene. Vertical lines indicate additional lanes removed for clarity. (D) Genome-wide occupancy of H3K27me3 and H3K27ac before and after wild-type H3F3A re-expression in H3K27M mutant cells. (E) Average distribution of H3K27me3 and H3K27ac peaks surrounding the transcription start site (TSS). (F) AFF4 promotor region before and after wild-type H3F3A re-expression demonstrates partial restoration of H3K27me3 peak, decrease in H3K27ac residue, and decrease in transcript by RNA-seq. (G) Decrease in AFF4 mRNA transcripts (left) and protein (right) following wild-type H3F3A re-expression. (H) Half-maximal inhibitory concentrations of atuveciclib across H3K27M+ DMG cultures, H3F3A wild-type (WT) pediatric glioblastoma (GBM01), immortalized astrocytes (NHA), ﬁbroblasts (NIH 3T3), and epithelial cells (RPE-NEO). (I) Half-maximal inhibitory concentrations of atuveciclib in immortalized human astrocytes before and after H3K27M expression. Experiments in (C) and (G) (qPCR) and (F) (RNA-seq) were performed with n = 3 technical replicates. Experiments in (H) and (I) were performed with minimum n = 3 biological replicates.

Journal: Cell reports

Article Title: Super Elongation Complex as a Targetable Dependency in Diffuse Midline Glioma.

doi: 10.1016/j.celrep.2020.03.049

Figure Lengend Snippet: Figure 5. The H3K27M Mutation Alters Epigenetic Regulation of AFF4 Promoter (A) H3K27ac deposition at the AFF4 promoter in neuroepithelial stem cells, OPCs, and differentiated brain regions (identical reads and scale for each sample tract). (B) H3K27ac deposition at the AFF4 promoter in patient-derived DMG cultures (identical reads and scale for each sample tract). (C) AFF4 mRNA transcripts (left) and protein expression (right) in H3F3A wild-type HSJD-GBM01 and normal human astrocytes following lentiviral transduction of the H3K27M transgene. Vertical lines indicate additional lanes removed for clarity. (D) Genome-wide occupancy of H3K27me3 and H3K27ac before and after wild-type H3F3A re-expression in H3K27M mutant cells. (E) Average distribution of H3K27me3 and H3K27ac peaks surrounding the transcription start site (TSS). (F) AFF4 promotor region before and after wild-type H3F3A re-expression demonstrates partial restoration of H3K27me3 peak, decrease in H3K27ac residue, and decrease in transcript by RNA-seq. (G) Decrease in AFF4 mRNA transcripts (left) and protein (right) following wild-type H3F3A re-expression. (H) Half-maximal inhibitory concentrations of atuveciclib across H3K27M+ DMG cultures, H3F3A wild-type (WT) pediatric glioblastoma (GBM01), immortalized astrocytes (NHA), ﬁbroblasts (NIH 3T3), and epithelial cells (RPE-NEO). (I) Half-maximal inhibitory concentrations of atuveciclib in immortalized human astrocytes before and after H3K27M expression. Experiments in (C) and (G) (qPCR) and (F) (RNA-seq) were performed with n = 3 technical replicates. Experiments in (H) and (I) were performed with minimum n = 3 biological replicates.

Techniques: Mutagenesis, Derivative Assay, Expressing, Transduction, Genome Wide, Residue, RNA Sequencing

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