antibodies against internal epitopes  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: Dual Function of Histone H3 Lysine 36 Methyltransferase ASH1 in Regulation of Hox Gene Expression

doi: 10.1371/journal.pone.0028171

Figure Lengend Snippet: (A) Quantitative RT-PCR analysis of HoxB and HoxC gene expression in K562 cells. Expression levels are normalized against average of K562 cells transfected with scrambled oligonucleotides. ASH1 knockdown oligonucleotides (si6925) causes reduction of ASH1 and several HoxB and HoxC genes, whereas β-actin is not affected. Values are average of triplicates of K562 samples and error bars denote standard deviation. (B) Effects of ASH1 knockdown on erythroid and monocytic marker gene expression in K562 cells. ASH1 knockdown causes increased expression of the ε-globin gene and decreased expression of GPIIb and GPIIIa genes. Transcription factors required for globin gene expression such as GATA1 and ELF1 are not affected. Data represent triplicate K562 samples and error bars indicate standard deviation. (C–D) Chromatin immunoprecipitation analysis of ASH1 in the HoxC8 (C) and HoxB6 (D) genes of K562 cells using antibodies against an internal epitope of ASH1. Quantitative PCR signals are shown by proportion to those of input DNA. GAPDH promoter is a negative control to which ASH1 does not bind. ASH1 is associated with promoter and coding regions of HoxC8 and HoxB6 genes. Values are average of triplicates of independent ChIP samples and indicate semi quantitative PCR signals relative to input DNA. Statistically significant differences (Student's t-test) are indicated by asterisks (*: p-value < 0.05, **: p-value < 0.01).

Article Snippet: Antibodies against C-terminal end of ASH1 or internal epitope of ASH1 (Santa-Cruz) were added and immunoprecipitated with Protein G-agarose coated with sermon sperm DNA (Clontech).

Techniques: Quantitative RT-PCR, Gene Expression, Expressing, Transfection, Knockdown, Standard Deviation, Marker, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

Journal: PLoS ONE

Article Title: Dual Function of Histone H3 Lysine 36 Methyltransferase ASH1 in Regulation of Hox Gene Expression

doi: 10.1371/journal.pone.0028171

Figure Lengend Snippet: (A) Luciferase reporter constructs harbouring human HoxC8 (−600 to +186) and HoxA9 (−637 to +56) promoters. (B) Activation of HoxC8-firefly luciferase and CMV-renilla luciferase reporters by MLL1, ASH1, or both together in HeLa cells. HoxC8-specific effects are shown by C8/CMV ratios. Values are average of triplicates of independently transfected HeLa cells and error bars denote standard deviation. (C) Similar to (B) but using HoxA9-firefly luciferase vector and ASH1 mutants including catalytically inactive ASH1 (H2113K) which induces twice as strong activation of HoxA9 (but not CMV) promoter as wild type ASH1. ASH1 1851-2694 that consists of the C-terminal region has significant activity towards HoxA9. ASH1 Δ216-1283 lacking a part of the N-terminal region can be detected by Western blot and has the same transactivation potential as wild type ASH1.

Article Snippet: Antibodies against C-terminal end of ASH1 or internal epitope of ASH1 (Santa-Cruz) were added and immunoprecipitated with Protein G-agarose coated with sermon sperm DNA (Clontech).

Techniques: Luciferase, Construct, Activation Assay, Transfection, Standard Deviation, Plasmid Preparation, Activity Assay, Western Blot

Journal: PLoS ONE

Article Title: Dual Function of Histone H3 Lysine 36 Methyltransferase ASH1 in Regulation of Hox Gene Expression

doi: 10.1371/journal.pone.0028171

Figure Lengend Snippet: (A) Effect of ASH1 and its mutants on endogenous HoxC8 (closed bars) and HoxB6 (open bars) genes in K562 cells. Catalytically inactive ASH1 H2113K augments endogenous Hox genes. Data represent triplicate K562 samples and error bars indicate standard deviation. Endogenous HoxC8 activation by ASH1 H2113K is statistically significant (*: p-value < 0.05). (B) Top: a reporter construct harbouring GAL4 binding sequences (x4) and c-fos promoter. Bottom: Expression vectors carrying GAL4-DNA binding domain (DBD), DBD fused to wild type ASH1 SET domain (DBD-ASH1), or DBD fused to catalytically inactive ASH1 SET domain (DBD-H2113K) were co-transfected with the reporter into HeLa cells. Wild type ASH1 SET domain is approximately 3-fold less efficient than catalytically inactive ASH1 SET domain, suggesting that K36 methylation attenuates transcription. Data represent triplicate HeLa samples and error bars indicate standard deviation. Differences between DBD and DBD-ASH1 and between DBD-ASH1 and DBD-H2113K were statistically significant (*: p-value < 0.01).

Article Snippet: Antibodies against C-terminal end of ASH1 or internal epitope of ASH1 (Santa-Cruz) were added and immunoprecipitated with Protein G-agarose coated with sermon sperm DNA (Clontech).

Techniques: Standard Deviation, Activation Assay, Construct, Binding Assay, Expressing, Transfection, Methylation

Journal: PLoS ONE

Article Title: Dual Function of Histone H3 Lysine 36 Methyltransferase ASH1 in Regulation of Hox Gene Expression

doi: 10.1371/journal.pone.0028171

Figure Lengend Snippet: Representative FACS plots of peripheral blood from mice transplanted with control GFP or knockdown vectors for ASH1 or MLL1. Horizontal axis represent CD45.1 (donor allotype) or CD45.2 (recipient allotype) and vertical axis indicates lineage markers: CD11b (macrophages), Gr-1 (granulocytes), CD19 (B cells), TCRβ (T cells), and TER119 (erythrocytes).

Article Snippet: Antibodies against C-terminal end of ASH1 or internal epitope of ASH1 (Santa-Cruz) were added and immunoprecipitated with Protein G-agarose coated with sermon sperm DNA (Clontech).

Techniques: Control, Knockdown