anti e2f1 rabbit polyclonal antibody  (Cell Signaling Technology Inc)

Journal: STAR Protocols

Article Title: Optimized lentiviral vector transduction of adherent cells and analysis in sulforhodamine B proliferation and chromatin immunoprecipitation assays

doi: 10.1016/j.xpro.2023.102109

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Article Snippet: Rabbit polyclonal anti-E2F1 , Cell Signaling Technology , CAT#3742S.

Techniques: Western Blot, Virus, Bacteria, Recombinant, Protease Inhibitor, Magnetic Beads, Plasmid Preparation, Control, Software, Blocking Assay, Inverted Microscopy, Transferring, Spectrophotometry, Hood

Journal: The Journal of Cell Biology

Article Title: Active RB causes visible changes in nuclear organization

doi: 10.1083/jcb.202102144

Figure Lengend Snippet: Investigation of the role of DP1, EZH2, HDAC, TOPIIα, and TOPIIβ in RB - mediated dispersion. (A) Western blots of WT and ΔCDK-RB RPE cells expressing GFP or DNDP1. MW, molecular weight. (B) E2F ChIP-qPCR and RB1 ChIP-qPCR showing that expression of DNDP1 decreased E2F1 and RB1 binding to sites in the MCM3, MCM4 promoters, compared with cells expressing only GFP. Average scores from three technical replicates were calculated per sample and per epitope. Holm–Sidak multiple t test was performed, and asterisks denote P values. Scale bar = 25 µm. (C) Effect of DNDP1 expression on cell cycle profile of the cells in A. Profile shows G1, S, and G2 cells in the different samples. DNDP1 expression in ΔCDK-RB cells interfered with G1 arrest and increased the percentage of cells in S and G2. (D) Effect of DNDP1 expression on dispersion. Quantitation of mean skeleton dot lengths after DNDP1 expression in WT RPE cells. Expression of DNDP1 in WT RPE does not cause a significant increase in mean skeleton dot length when compared with WT RPE cells or GFP-expressing WT RPE cells. (E) Mean skeleton dot lengths (chromosome 7 α-satellite probe) after WT and ΔCDK-RB RPE cells were treated with EZH2 inhibitor. (F) Western blot for WT RPE cells treated with DMSO or EZH2 inhibitor (Inh.). Note that treatment with EZH2 inhibitor reduces H3K27 trimethylation levels. (G) Western blot for WT contact inhibited (C.I.) RPE and ΔCDK-RB treated with TSA for 72 h. H4 acetylation in both WT C.I. and ΔCDK-RB cells increases after TSA treatment. (H and I) Western blot for WT RPE and ΔCDK-RB transfected with control and TOPIIα (H) or TOPIIβ (I) siRNAs. siRNA-mediated knockdown of TOPIIα reduced the levels of the appropriate endogenous protein in WT and ΔCDK-RB cells. We note that TOPIIα was expressed at lower levels in cells expressing ΔCDK-RB compared with WT RPE cells. TOPIIβ siRNA-mediated knockdown causes complete loss of endogenous TOPIIβ in both WT and ΔCDK-RB cells. It was also observed that TOPIIβ levels were lower in ΔCDK-RB, compared with WT. (J) Mean skeleton dot lengths (chromosome 7 α-satellite probe) after WT and ΔCDK-RB RPE cells were treated with control and TOPIIα siRNAs. Numbers of foci quantified for each sample ( n ) are as follows (in the order they appear on the bar graphs): D, n = 29, 48, 40, 29; E, n = 37, 82, 19, 58; J, n = 103, 75, 75, 56. Error bars are SEM. Nonparametric two-tailed Mann–Whitney U test was performed for pairs of samples indicated on graphs, and asterisks denote P values. ns, P > 0.05; ***, P ≤ 0.001. Source data are available for this figure: . Dashed lines indicate the cutoffs for defining the categories compact, diffused, and dispersed for chromosome 7 α-satellite.

Article Snippet: Solubilized chromatin was immunoprecipitated with FLAG rabbit monoclonal antibody (14793; Cell Signaling Technologies) or E2F1 rabbit polyclonal antibody (3742; Cell Signaling Technologies) overnight at 4°C.

Techniques: Dispersion, Western Blot, Expressing, Molecular Weight, ChIP-qPCR, Binding Assay, Quantitation Assay, Transfection, Control, Knockdown, Two Tailed Test, MANN-WHITNEY

Journal: Cancer letters

Article Title: Identification of a novel catalytic inhibitor of topoisomerase II alpha that engages distinct mechanisms in p53 wt or p53 -/- cells to trigger G2/M arrest and senescence.

doi: 10.1016/j.canlet.2021.11.025

Figure Lengend Snippet: Fig. 7. E2F1 is stabilized in response to MPO. (A) HCT116 p53WT and (B) p53−/−cells were treated with MPO (20 μM) for 24–96 h and lysates from the nuclear and cytosolic fractions were subjected to Western blotting analysis using anti-E2F1 and anti-β actin antibodies. (C) HCT p53WT cells were treated with MPO (20 μM) for 24 h in the presence or absence of cycloheximide (CHX) (2.5–5 μg/ml) or actinomycin D (Act. D) (0.5 μg/ml) and cell lysates were subjected to Western blotting analysis and probed with anti-E2F1 or anti-β actin antibodies.

Article Snippet: The following antibodies were used in the study: Mouse monoclonal β-actin (Sigma Aldrich Co., St. Louis, MO), mouse monoclonal p53, p21, topo IIα, (BD Pharmingen, San Diego, CA, USA), mouse monoclonal phospho-p53(ser15), phospho-ATM(ser1981) (Cell Signaling Technology Inc., Danvers, MA), mouse monoclonal ATM, Chk2, Chk1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), mouse monoclonal phosphohistone H2AX(ser139), clone JBW301 (Upstate Biotechnology Inc., Lake Placid, NY), rabbit monoclonal phospho-Chk2(thr68), phospho-Chk1 (ser345) (Cell Signaling Technology Inc., Danvers, MA), rabbit polyclonal E2F1 (Cell Signaling Technology Inc., Danvers, MA), rabbit polyclonal topo IIα (TopoGEN, Inc., Columbus, OH), rabbit polyclonal ATR (Calbiochem, San Diego, CA), goat anti-mouse IgG HRP conjugated and goat anti-rabbit IgG HRP conjugated secondary antibodies (Pierce Chemical Co., Rockford, IL).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Differential regulation of mammalian and avian ATOH1 by E2F1 and its implication for hair cell regeneration in the inner ear

doi: 10.1038/s41598-021-98816-w

Figure Lengend Snippet: Effect of the E2F transcription factors on the mouse and chick Atoh1 enhancers and putative enhancer C. ( A ) Schematic representation of the luciferase constructs containing the mouse or chick Atoh1 conserved regions cloned into the luciferase vector pGL4.23 . ( B ) Dose–response effects on the Atoh1 luciferase constructs of transfecting increasing amounts of an E2F1 expression construct or mutant E2F1 in the UB/OC-2 cell line. ( C ) Effect of E2F2, E2F3 and E2F4 on the activity of the Atoh1 luciferase constructs. Experiments were conducted in triplicate in two separate assays with different DNA preparations for each plasmid in each assay (n = 6). Relative luciferase values are expressed relative to activity of the reporters in the absence of E2F. Student t -test was conducted comparing each condition with 0 ng of the corresponding E2F expression construct. (* p < 0.05; ** p < 0.01; # p < 10 –5 ; ## p < 10 –10 ). Error bars represent the s.e.m (n = 6).

Article Snippet: Immunohistochemistry on chick tissues was performed as described previously with the following antibodies: chicken anti-peptide polyclonal against mouse Atoh1 (used at 1:5000) kindly supplied by Matthew Kelley ; rabbit polyclonal E2F1 (against the N terminal of E2F1; P100821_P050) from Aviva Systems Biology used at 1:200; mouse monoclonal anti-SOX2 (BD Pharmingen) used at 1:200.

Techniques: Luciferase, Construct, Clone Assay, Plasmid Preparation, Expressing, Mutagenesis, Activity Assay

Journal: Scientific Reports

Article Title: Differential regulation of mammalian and avian ATOH1 by E2F1 and its implication for hair cell regeneration in the inner ear

doi: 10.1038/s41598-021-98816-w

Figure Lengend Snippet: Identification and verification of E2F recognition elements in the chick Atoh1 conserved elements. ( A ) Schematic diagram showing the location of nine putative E2F binding sites predicted in the chick Atoh1 conserved regions by MatInspector software (labelled as S1–S9) Numbers indicate the position of the enhancers in base pairs. ( B ) Immunofluoresence detection of endogenous expression of ATOH1 (green) and E2F1 (magenta) proteins in UB/OC-2 cells co-stained with DAPI (blue). ( C ) and ( D ): EMSA analysis performed using a radiolabelled E2F1 consensus sequence, containing a functional E2F1 binding site incubated alone (lane 1), with untransfected nuclear extracts or with nuclear extracts transfected with E2F1 and DP1 expression vectors as indicated. Competition assays were performed with 500 ng of non-radiolabelled competitors (consensus E2F1, lane 3 in both ( C ) and ( D ), and lane 9 in ( C ); mutant E2F1, lane 4 in ( C ) and ( D ), and lane 10 in ( C ); non-specific (N/S) probe, lane 5 and 11 in ( C ); sites S1 to S9 as described in ( A ), lanes 7–15 in ( D ). Lane 6 and 12 show a supershift assay with rabbit polyclonal anti-E2F1, pre-incubated with extracts before the binding assay.

Article Snippet: Immunohistochemistry on chick tissues was performed as described previously with the following antibodies: chicken anti-peptide polyclonal against mouse Atoh1 (used at 1:5000) kindly supplied by Matthew Kelley ; rabbit polyclonal E2F1 (against the N terminal of E2F1; P100821_P050) from Aviva Systems Biology used at 1:200; mouse monoclonal anti-SOX2 (BD Pharmingen) used at 1:200.

Techniques: Binding Assay, Software, Expressing, Staining, Sequencing, Functional Assay, Incubation, Transfection, Mutagenesis

Journal: Scientific Reports

Article Title: Differential regulation of mammalian and avian ATOH1 by E2F1 and its implication for hair cell regeneration in the inner ear

doi: 10.1038/s41598-021-98816-w

Figure Lengend Snippet: The effect of site directed mutagenesis of the E2F1 putative sites 2 and 6 on E2F1 mediated activation of the Atoh1 regulatory region. ( A ) Consensus seuqeunce of the E2F1 binding site described by MatInspector, Genomatix. Cross species alignment of sites 2 and site 6 in the Atoh1 regulatory regions. Conserved nucleotides within the core sequence of site 2 and site 6 are shown in red. ( B ) Diagram showing the sequence and position of putative E2F sites 2 and 6 which both contain overlapping predicted binding sites for E2F1 and E2F4. Point mutations were introduced in the core GC nucleotides, replaced with TT (marked in bold and with asterisks). ( C ) Luciferase assays show a reduced response of the chABC-s2mut (* p < 0.05) and the chABC-s6mut (*** p < 0.001) to E2F1 transfection in comparison with the wild type luciferase construct ( chABC-wt ). Each experiment was conducted in triplicate in two separate assays with different DNA preparations. Student t-test was conducted.

Article Snippet: Immunohistochemistry on chick tissues was performed as described previously with the following antibodies: chicken anti-peptide polyclonal against mouse Atoh1 (used at 1:5000) kindly supplied by Matthew Kelley ; rabbit polyclonal E2F1 (against the N terminal of E2F1; P100821_P050) from Aviva Systems Biology used at 1:200; mouse monoclonal anti-SOX2 (BD Pharmingen) used at 1:200.

Techniques: Mutagenesis, Activation Assay, Binding Assay, Sequencing, Luciferase, Transfection, Construct

Journal: Scientific Reports

Article Title: Differential regulation of mammalian and avian ATOH1 by E2F1 and its implication for hair cell regeneration in the inner ear

doi: 10.1038/s41598-021-98816-w

Figure Lengend Snippet: E2F1 expression during HC regeneration in the chick BP. ( A ) Schematic representation of the experimental timeline of organotypic cultures of E18 chick BPs cultured in DMEM and 1% FBS for 2 days with 78 µM streptomycin (Strep) followed an additional 3 or 6 days in vitro (DIV) in the same media without streptomycin. The arrows indicate when the tissue was harvested. ( B ) Immunofluorescence of BPs showing the expression of E2F1 and ATOH1 in HCs (labelled with Phalloidin) and SCs (labelled with SOX2). Cultures maintained for 2 days in media with Strep show ATOH1 re-activation in some SCs whereas a downregulation of nuclear E2F1 expression is observed in comparison to control cultures maintained in DMEM (hollow arrowheads in zoom in areas in row (I) and (IV); scale bar: 10 µm. In BPs cultured for 3 additional days in streptomycin-free media (2d Strep + 3 DIV), nuclear E2F1 expression is observed in SCs (hollow arrowheads in zoom in areas in row II); scale bar: 10 µm). New HCs labelled with Phalloidin are formed in cultures maintained for 6 days in streptomycin-free media after drug treatment (2d Strep + 6 days DIV). In these cultures, ATOH1 is downregulated in SCs labelled with SOX2 whereas E2F1 is expressed in SCs and HCs (arrowheads in row III in E2F1 panel). Control experiments were conducted in parallel in DMEM and at the same timings plus additional days of in vitro (DIV). Technical replicates were three for each experiment with at least three biological samples.

Article Snippet: Immunohistochemistry on chick tissues was performed as described previously with the following antibodies: chicken anti-peptide polyclonal against mouse Atoh1 (used at 1:5000) kindly supplied by Matthew Kelley ; rabbit polyclonal E2F1 (against the N terminal of E2F1; P100821_P050) from Aviva Systems Biology used at 1:200; mouse monoclonal anti-SOX2 (BD Pharmingen) used at 1:200.

Techniques: Expressing, Cell Culture, In Vitro, Immunofluorescence, Activation Assay

Journal: Scientific Reports

Article Title: Differential regulation of mammalian and avian ATOH1 by E2F1 and its implication for hair cell regeneration in the inner ear

doi: 10.1038/s41598-021-98816-w

Figure Lengend Snippet: Working hypothesis on the role of E2F1 on the control of Atoh1 . Upon HC damage, cytoplasmic accumulation of E2F1 in SCs may occur in response to damage as an early response. Cytoplasmic E2F1 may be involved in apoptotic functions or controlling translation of proteins as an RNA-binding protein. A later response might be triggered to activate regeneration via cell division by shifting E2F1 protein into the nucleus of SCs. In the nucleus and upon phosphorylation of the Rb protein, E2F1 promotes re-entry of post-mitotic SCs into the cell cycle (from G 0 phase to G 1 phase). Nuclear E2F1 simultaneously induces transcription of chick Atoh1 via a regulatory element in putative enhancer C resulting in re-activation of the expression of chick ATOH1. This mechanism could be responsible for the spontaneous HC regeneration in avian species. In contrast, mouse Atoh1 gene is not responsive to E2F1 and therefore limits HC regeneration in mammalian species.

Article Snippet: Immunohistochemistry on chick tissues was performed as described previously with the following antibodies: chicken anti-peptide polyclonal against mouse Atoh1 (used at 1:5000) kindly supplied by Matthew Kelley ; rabbit polyclonal E2F1 (against the N terminal of E2F1; P100821_P050) from Aviva Systems Biology used at 1:200; mouse monoclonal anti-SOX2 (BD Pharmingen) used at 1:200.

Techniques: RNA Binding Assay, Activation Assay, Expressing