Anti-E2F1 antibody [KH95]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E2F1 antibody [KH95] (AB4070)

ab4070 staining human tonsil by IHC-P.

• WB

Lab

Western blot - Anti-E2F1 antibody [KH95] (AB4070)

Western blot : Anti-E2F1 antibody [KH95] (ab4070) staining at 1/500 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab4070 was shown to bind specifically to E2F1. A band was observed at 47 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in E2F1 knockout cell line. To generate this image, wild-type and E2F1 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-E2F1 antibody [KH95] (ab4070) at 1/500 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

E2F1 knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type HEK-293 ab259780 cell lysate at 20 µg

Lane 4:

E2F1 knockout HEK-293 <a href='/en-us/products/cell-lines/human-e2f1-knockout-hek-293-cell-line-ab269502'>ab269502</a> cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

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• WB

Lab

Western blot - Anti-E2F1 antibody [KH95] (AB4070)

Lanes 1 - 5 : Merged signal (red and green). Green - ab4070 observed at 55 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37 kDa.

ab4070 was shown to react with E2F1 in wild-type HEK-293 cells in Western blot with loss of signal observed in E2F1 knockout sample. Wild-type HEK-293 and E2F1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab4070 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at a 1 in 500 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-E2F1 antibody [KH95] (ab4070) at 1/500 dilution

Lane 1:

Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

E2F1 knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Western blot - Human E2F1 knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-e2f1-knockout-hek-293-cell-line-ab269502'>ab269502</a>)

Lane 3:

Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate at 20 µg

Lane 5:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Predicted band size: 47 kDa

Observed band size: 55 kDa

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• WB

CiteAb

Western blot - Anti-E2F1 antibody [KH95] (AB4070)

Western Blotting using Anti-E2F1 antibody [KH95]Anti-E2F1 antibody [KH95], ab4070. Publication image from Meng, X. et al., 2020, Nat Commun, 32001707. Legend direct from paper.

BIP-MPC-NP restrains E2F1-mediated DNA damage repair modules via the inhibitory effect of TTP.a E2F1 binding sites within a region spanning ± 3 kb around TSS in the whole genome. b The signal peaks located in the promoter regions of CHEK1, CHEK2, RAD50, RAD51 and TP53 in E2F1 ChIP-seq data and the binding sites of E2F1 were predicted on JASPAR datasets. The agarose gel electrophoresis displayed the enrichments of E2F1 in the promoter regions of CHEK1, CHEK2, RAD50, RAD51 and TP53 of LN229R. c The luciferase reporter assay displayed the E2F1 transcriptional activity in the promoter regions of CHEK1, CHEK2, RAD50, RAD51 and TP53 in LN229R (n = 3). d The TTP protein expression and E2F1 mRNA expression in LN229R, U87MGR and HG9R treated with MPC-NP or BIP-MPC-NP. e The inhibitory effect of TTP on wild-type or mutant-type ARE motifs within the E2F1 mRNA 3′-UTR in LN229R cells with MPC-NP or BIP-MPC-NP treatments (n = 3). The error bars in c and e represent the S.D. of three measurements. P value is determined by Student’s t-test. Significant results are presented as NS non-significant, *P < 0.05, **P < 0.01, ***P < 0.001.

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• WB

CiteAb

Western blot - Anti-E2F1 antibody [KH95] (AB4070)

Western Blotting using Anti-E2F1 antibody [KH95]Anti-E2F1 antibody [KH95], ab4070. Publication image from Kanakkanthara, A. et al., 2019, Nat Commun, 31604914. Legend direct from paper.

ZC3H18 binds the BRCA1 promoter and inhibits E2F1 binding. a Schematic of the BRCA1 proximal promoter with E2FA and E2FB sites indicated. Nucleotide sequences of the DNA probes used in the electrophoretic mobility shift assays (EMSA). E2FA and E2FB mutation sites are indicated in open rectangles. b EMSA with purified recombinant SFB-ZC3H18 using BRCA1 promoter probe with wild-type sequence (E2FA/BWT) or mutations in the E2FA site (E2FδA), the E2FB site (E2FδB), or both E2F sites (E2FδA/B). A probe with randomly shuffled sequences was used as negative control. For supershift assays, an anti-S-Tag monoclonal antibody, which binds the SFB tag in SFB-ZC3H18, was used. c, d ZC3H18 and E2F4 co-occupy the endogenous BRCA1 promoter. Sequential ChiP (ChIP-Re-ChIP) assays in OVCAR-8 cells using anti-ZC3H18 antibody for primary ChIP and anti-E2F4 antibody for secondary ChIP (c) and using anti-E2F4 antibody for primary ChIP and anti-ZC3H18 antibody for secondary ChIP (d). e EMSA with purified recombinant SFB-ZC3H18 and SFB-E2F1 using BRCA1 promoter probe with mutated E2FB site (E2FδB). f EMSA with purified SFB-E2F4 and SFB-E2F1 using BRCA1 promoter probe with mutated E2FA site (E2FδA). The images of EMSA in b, e, and f are representative of three independent experiments that gave similar results. Data in c and d are means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test

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• WB

CiteAb

Western blot - Anti-E2F1 antibody [KH95] (AB4070)

Western Blotting using Anti-E2F1 antibody [KH95]Anti-E2F1 antibody [KH95], ab4070. Publication image from Kanakkanthara, A. et al., 2019, Nat Commun, 31604914. Legend direct from paper.

ZC3H18 occupies the BRCA1 promoter and regulates E2F1. a OVCAR-8, OVCAR-5, and PEA1 cells were processed for ChIP assays using anti-ZC3H18 or IgG control antibodies and primers specific for the BRCA1 promoter. b OVCAR-8 cells transiently expressing SFB-ZC3H18 were processed for ChIP assays using FLAG or IgG control antibodies. c, d ChIP assays showing increased occupancy of E2F1 on the BRCA1 promoter when ZC3H18 is depleted. OVCAR-8 (c) and PEA1 (d) cells transfected with control luciferase (Luc) or ZC3H18 siRNAs (siZC3) were processed for ChIP using anti-E2F1 or IgG control antibodies 48 h after transfection. e E2F1 depletion does not affect BRCA1 levels. OVCAR-8 cells were transfected with control luciferase (Luc) or two independent E2F1 siRNAs and analyzed for BRCA1, E2F1, and HSP90 levels by immunoblotting 48 h later. f DNMT1 occupancy on the BRCA1 promoter requires E2F1. OVCAR-8 cells transfected with Luc, E2F1, or ZC3H18 siRNA with our without E2F1 siRNAs were processed for ChIP with DNMT1 or control IgG antibodies 48 h after transfection. g Depletion of E2F1 restores BRCA1 expression when ZC3H18 is depleted. OVCAR-8 cells transfected with Luc, E2F1, or ZC3H18 siRNAs with our without E2F1 siRNAs were analyzed for BRCA1 mRNA levels by qRT-PCR. BRCA1 mRNA levels were normalized to GAPDH mRNA levels. Data in a–g are means ± SEM from three independent experiments. **p < 0.01, ***p < 0.001, unpaired Student’s t-test. Representative immunoblots in e are provided from three independent experiments. Unprocessed blots are provided in Source data file

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