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anti e2f1 rabbit polyclonal antibody (Cell Signaling Technology Inc)

Name: anti e2f1 rabbit polyclonal antibody
Brand: Cell Signaling Technology Inc
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Cell Signaling Technology Inc anti e2f1 rabbit polyclonal antibody
Anti E2f1 Rabbit Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 630 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti e2f1 rabbit polyclonal antibody/product/Cell Signaling Technology Inc
Average 96 stars, based on 630 article reviews

anti e2f1 rabbit polyclonal antibody - by Bioz Stars, 2026-02

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ARRB1 controls cell proliferation and apoptosis in granule cell progenitors (GCPs) via E2F1 acetylation. (A) Murine cerebellum Western blot. Representative images of endogenous GLI1 and ARRB1 in murine cerebella at different days (2, 5, 7, 15) of cerebellar development. GAPDH: loading control. (B–G) GCPs experiments. Shh was added to cultures of murine cerebellar GCPs that had or had not undergone siRNA-mediated silencing of ARRB1 (siArrb1). After 48 h of Shh stimulation, GCPs were assayed for: (B) Gli1 mRNA and protein levels (as a read-out of Shh signaling activity), and ARRB1 protein level; ACTIN as loading control; (C) proliferation reflected by bromodeoxyuridine (BrdU) uptake; (D) left: p27 mRNA levels ( (D) right and : Overexpression data are consistent); (E) differentiation reflected by β III tubulin mRNA levels; (F) apoptosis evaluated by TUNEL assay, and (G) ARRB1, E2F1, E2F1-ac, cleaved CASPASE-3 protein expression levels. ACTIN and HISTONE H3: loading controls. For Western blot, densitometry values are shown below the blots and densitometric graphs are presented in . Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001.

Journal: Frontiers in Cell and Developmental Biology

Article Title: β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum

doi: 10.3389/fcell.2023.990711

Figure Lengend Snippet: ARRB1 controls cell proliferation and apoptosis in granule cell progenitors (GCPs) via E2F1 acetylation. (A) Murine cerebellum Western blot. Representative images of endogenous GLI1 and ARRB1 in murine cerebella at different days (2, 5, 7, 15) of cerebellar development. GAPDH: loading control. (B–G) GCPs experiments. Shh was added to cultures of murine cerebellar GCPs that had or had not undergone siRNA-mediated silencing of ARRB1 (siArrb1). After 48 h of Shh stimulation, GCPs were assayed for: (B) Gli1 mRNA and protein levels (as a read-out of Shh signaling activity), and ARRB1 protein level; ACTIN as loading control; (C) proliferation reflected by bromodeoxyuridine (BrdU) uptake; (D) left: p27 mRNA levels ( (D) right and : Overexpression data are consistent); (E) differentiation reflected by β III tubulin mRNA levels; (F) apoptosis evaluated by TUNEL assay, and (G) ARRB1, E2F1, E2F1-ac, cleaved CASPASE-3 protein expression levels. ACTIN and HISTONE H3: loading controls. For Western blot, densitometry values are shown below the blots and densitometric graphs are presented in . Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The following antibodies were used: anti-GLI1 (L42B10, Cell Signaling), anti-CASPASE-3 (D3R6Y, Cell Signaling), anti-β-ARRESTIN1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-ACTIN I-19 (sc-1616; Santa Cruz Biotechnology), anti-E2F1 C-20 (sc-193; Santa Cruz Biotechnology), anti-E2F1 (acetyl K120/K125) (AP10555SU-N, Acris Antibodies); anti-ZIC1 (ab72694; Abcam); anti-PARP p85 Fragment (G7342; Promega), anti-SP1 1C6 (sc-420X; Santa Cruz Biotechnology), anti-PCNA (2586; Cell signaling), anti-H3 (ab 1971, Abcam), and anti-GAPDH (ab8245; Abcam).

Techniques: Western Blot, Control, Activity Assay, Over Expression, TUNEL Assay, Expressing

E2F1-ac targets’ transcripts after ARRB1 modulation. (A,B) Expression of pro-apoptotic E2F1-ac target genes ( Trp73, Casp3 and Casp7 ) in GCPs that had or had not undergone siArrb1 (black) or over-expression of Arrb1 (Arrb1-HA) (orange). (C,D) Expression of proliferative (grey) and epithelial mesenchymal transition (violet) E2f1-ac target genes ( Cdc25a, Birc5, Tyms, Zeb1, Zeb2, Vim and Fn1 ) in GCPs that had or had not undergone siArrb1. Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001. (E) IHC staining for ARRB1 and P27 in representative mouse cerebellum sections at several differentiation stages (p2, p4, p7, p10, p15) (upper panels). Magnification ×10; insets ×40. Scale bar, 250 µm. CASPASE-3 and CDC25A indexes (expressed as percentage) calculated as DAB positive cells of total number of cells (bottom panels). Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.001 vs. p10. # p < 0.05; ## p < 0.01 vs. p15.

Journal: Frontiers in Cell and Developmental Biology

Article Title: β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum

doi: 10.3389/fcell.2023.990711

Figure Lengend Snippet: E2F1-ac targets’ transcripts after ARRB1 modulation. (A,B) Expression of pro-apoptotic E2F1-ac target genes ( Trp73, Casp3 and Casp7 ) in GCPs that had or had not undergone siArrb1 (black) or over-expression of Arrb1 (Arrb1-HA) (orange). (C,D) Expression of proliferative (grey) and epithelial mesenchymal transition (violet) E2f1-ac target genes ( Cdc25a, Birc5, Tyms, Zeb1, Zeb2, Vim and Fn1 ) in GCPs that had or had not undergone siArrb1. Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001. (E) IHC staining for ARRB1 and P27 in representative mouse cerebellum sections at several differentiation stages (p2, p4, p7, p10, p15) (upper panels). Magnification ×10; insets ×40. Scale bar, 250 µm. CASPASE-3 and CDC25A indexes (expressed as percentage) calculated as DAB positive cells of total number of cells (bottom panels). Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.001 vs. p10. # p < 0.05; ## p < 0.01 vs. p15.

Techniques: Expressing, Over Expression, Immunohistochemistry

ARRB1-E2F1 complex direct regulates the expression of E2F1-ac pro-apoptotic targets. (A,B) qPCR-ChIP assay of ARRB1 in GCPs stimulated or not with Shh. Immunoprecipitation with IgG was performed as control. Eluted DNA was amplified by qPCR using primers specific for the regulatory region of the indicated genes. Actin and Gapdh (not shown) were used as endogenous non-enriched regions. qPCR data are presented as percentage of ChIP input controls. Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001.

Journal: Frontiers in Cell and Developmental Biology

Article Title: β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum

doi: 10.3389/fcell.2023.990711

Figure Lengend Snippet: ARRB1-E2F1 complex direct regulates the expression of E2F1-ac pro-apoptotic targets. (A,B) qPCR-ChIP assay of ARRB1 in GCPs stimulated or not with Shh. Immunoprecipitation with IgG was performed as control. Eluted DNA was amplified by qPCR using primers specific for the regulatory region of the indicated genes. Actin and Gapdh (not shown) were used as endogenous non-enriched regions. qPCR data are presented as percentage of ChIP input controls. Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001.

Techniques: Expressing, Immunoprecipitation, Control, Amplification

ARRB1 controls NSCs apoptosis via E2F1 acetylation. (A) Effects of Arrb1-HA overexpression and mock transfection (control) of NSCs on ARRB1 and E2F1-ac protein levels (Western blot assay-left) and Trp73 and Cdc25a mRNA levels (right). * p < 0.05 vs. mock transfected cells (Mock). GAPDH: loading control for Western blot. (B) Western Blot analysis of endogenous ARRB1, E2F1, and E2F1-ac expression in NSCs cultured in SCM (0 h) and after 8–24 h culture in DFM. GAPDH: loading control. Effects of si-Arrb1 or scrambled control (siCtr) on (C) Trp73 and Cdc25a mRNA levels (left) and ARRB1 protein levels (Western blot assay-right) in NSCs cultured in SCM (0 h) and after 18 h culture in DFM. ACTIN: loading control for Western blot. For Western blot, densitometry values are shown below the blots and densitometric graphs are presented in . * p < 0.05 vs. scrambled control (siCtr).

Journal: Frontiers in Cell and Developmental Biology

Article Title: β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum

doi: 10.3389/fcell.2023.990711

Figure Lengend Snippet: ARRB1 controls NSCs apoptosis via E2F1 acetylation. (A) Effects of Arrb1-HA overexpression and mock transfection (control) of NSCs on ARRB1 and E2F1-ac protein levels (Western blot assay-left) and Trp73 and Cdc25a mRNA levels (right). * p < 0.05 vs. mock transfected cells (Mock). GAPDH: loading control for Western blot. (B) Western Blot analysis of endogenous ARRB1, E2F1, and E2F1-ac expression in NSCs cultured in SCM (0 h) and after 8–24 h culture in DFM. GAPDH: loading control. Effects of si-Arrb1 or scrambled control (siCtr) on (C) Trp73 and Cdc25a mRNA levels (left) and ARRB1 protein levels (Western blot assay-right) in NSCs cultured in SCM (0 h) and after 18 h culture in DFM. ACTIN: loading control for Western blot. For Western blot, densitometry values are shown below the blots and densitometric graphs are presented in . * p < 0.05 vs. scrambled control (siCtr).

Techniques: Over Expression, Transfection, Control, Western Blot, Expressing, Cell Culture

Schematic model of ARRB1/E2F1-ac functions in GCPs and NSCs. Overview of roles played by ARRB1/E2F1-ac in normal cerebellar development. (A) : Committed neuronal precursors (i.e., NSCs grown in DFM, GCPs). In our previous works, we identified miR-326 as a miRNA necessary for maturation of granule cell progenitors (GCPs) into mature granule cells . Moreover, this miRNA is integrated into the first intron of the Arrb1 gene and shares the same regulatory regions as its host gene. miR-326 also contributes to ARRB1 functions by blunting proliferative signals mediated by E2F1, Hedgehog, and Notch, and by promoting cell differentiation ( ; ; ; ). Committed neuronal precursors express ARRB1 and mir-326, which regulate their development at multiple levels. Shh signaling upregulates ARRB1 levels and promotes its translocation to the nucleus. There ARRB1, in complex with P300, induces acetylation of E2F1 (E2F1-ac), redirecting the transcription factors activity from survival/proliferative gene targets towards those that promote apoptosis ( Trp73, Caspases 3 and 7 ). Interacting with CREB and P300, ARRB1 upregulates the expression and nuclear accumulation of P27, which eventually blocks cell cycle progression. miR-326 favors neuronal cell differentiations by inhibiting multiple survival/proliferative signaling: E2F1, Hedgehog (Hh) and Notch via direct binding of the 3′-UTRs of E2f1, Smo, Gli2, Notch1 and Notch2 . (B) : In NSCs, non-expression of ARRB1 and miR-326 promotes cell proliferation, survival, and stemness by favoring non-acetylated E2F1 activity and active Hedgehog (Hh) and Notch signaling.

Journal: Frontiers in Cell and Developmental Biology

Article Title: β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum

doi: 10.3389/fcell.2023.990711

Figure Lengend Snippet: Schematic model of ARRB1/E2F1-ac functions in GCPs and NSCs. Overview of roles played by ARRB1/E2F1-ac in normal cerebellar development. (A) : Committed neuronal precursors (i.e., NSCs grown in DFM, GCPs). In our previous works, we identified miR-326 as a miRNA necessary for maturation of granule cell progenitors (GCPs) into mature granule cells . Moreover, this miRNA is integrated into the first intron of the Arrb1 gene and shares the same regulatory regions as its host gene. miR-326 also contributes to ARRB1 functions by blunting proliferative signals mediated by E2F1, Hedgehog, and Notch, and by promoting cell differentiation ( ; ; ; ). Committed neuronal precursors express ARRB1 and mir-326, which regulate their development at multiple levels. Shh signaling upregulates ARRB1 levels and promotes its translocation to the nucleus. There ARRB1, in complex with P300, induces acetylation of E2F1 (E2F1-ac), redirecting the transcription factors activity from survival/proliferative gene targets towards those that promote apoptosis ( Trp73, Caspases 3 and 7 ). Interacting with CREB and P300, ARRB1 upregulates the expression and nuclear accumulation of P27, which eventually blocks cell cycle progression. miR-326 favors neuronal cell differentiations by inhibiting multiple survival/proliferative signaling: E2F1, Hedgehog (Hh) and Notch via direct binding of the 3′-UTRs of E2f1, Smo, Gli2, Notch1 and Notch2 . (B) : In NSCs, non-expression of ARRB1 and miR-326 promotes cell proliferation, survival, and stemness by favoring non-acetylated E2F1 activity and active Hedgehog (Hh) and Notch signaling.

Techniques: Cell Differentiation, Translocation Assay, Activity Assay, Expressing, Binding Assay

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

Figure Lengend Snippet:

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

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

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

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).

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

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.

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.

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

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.

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.

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

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.

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.

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

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.

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.

Techniques: RNA Binding Assay, Activation Assay, Expressing

Sequences of shRNAs and primers used for reverse transcription-quantitative PCR.

Journal: Oncology Reports

Article Title: Nucleolar and spindle-associated protein 1 promotes non-small cell lung cancer progression and serves as an effector of myocyte enhancer factor 2D

doi: 10.3892/or.2020.7918

Figure Lengend Snippet: Sequences of shRNAs and primers used for reverse transcription-quantitative PCR.

Article Snippet: The membranes were incubated with 5% skimmed milk at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-NUSAP1 polyclonal antibody (1:100; cat. no. 12024-1-AP; ProteinTech Group, Inc.), rabbit anti-MEF2D polyclonal antibody (1:800; cat. no. ab32845; Abcam), mouse anti-GAPDH monoclonal antibody (1:20,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.), rabbit anti-β-catenin monoclonal antibody (1:9,000; cat. no. ab32572; Abcam), rabbit anti-c-Myc monoclonal antibody (1:600; cat. no. ab32072; Abcam), mouse anti-cyclin D1 monoclonal antibody (1:6,000; cat. no. 60186-1-Ig; ProteinTech Group, Inc.), rabbit anti-matrix metallopeptidase (MMP7) polyclonal antibody (1:500; cat. no. 10374-2-AP; ProteinTech Group, Inc.), rabbit anti-histone H3 polyclonal antibody (1:4,000; cat. no. 17168-1-AP; ProteinTech Group, Inc.), rabbit anti-ZEB1 polyclonal antibody (1:1,000; cat. no. 21544-1-AP; ProteinTech Group, Inc.) and rabbit anti-E2F1 polyclonal antibody (1:800; cat. no. 12171-1-AP; ProteinTech Group, Inc.).

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