Anti-MBD3 antibody [EPR9913] - ChIP Grade

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling MBD3 with purified ab157464 at 1/1000. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (ab150077). Nuclei counterstained with DAPI (blue).

Control : PBS only

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Intracellular flow cytometric analysis of permeabilized 293T cells labeling MBD3 with ab157464 at 1/10 dilution (red) compared to a rabbit IgG negative control (green).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Immunofluorescent analysis of HeLa cells labeling MBD3 with ab157464 at 1/100 dilution.

• ChIP

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ChIP - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Chromatin was prepared from HeLa cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab157464 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.

• ChIP

Unknown

ChIP - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Chromatin was prepared from NIH/3T3 cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab157464 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.

• WB

Lab

Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Lanes 1 - 4 : Merged signal (red and green). Green - ab157464 observed at 33 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab157464 was shown to recognize MBD3 when MBD3 knockout samples were used, along with additional cross-reactive bands. Wild-type and MBD3 knockout samples were subjected to SDS-PAGE. ab157464 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464) at 1/1000 dilution

Lanes 1 and 3:

Wild-type HAP1 cell lysate at 20 µg

Lanes 2 and 4:

MBD3 knockout HAP1 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed at 1/10000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed at 1/10000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

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• WB

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Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

All lanes:

Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

Fetal brain lysate at 10 µg

Lane 4:

Y79 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 33 kDa

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• WB

CiteAb

Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Western Blotting using Anti-MBD3 antibody [EPR9913] - ChIP Grade, ab157464. Publication image from Ma, C. et al., 2018, Mol Cancer, 29625565. Legend direct from paper.

Involvement of NuRD complex in the regulation of breast cancer cell senescence and suppression mediated by SALL1. a and b Transfection of mutated SALL1 (mSALL1, deleted the NuRD binding peptide motif of conserved 12-amino) in MCF-7 and E0771 cancer cells did not induce SA-β-Gal+ cell populations (in a) and promote cancer cell cycle arrest in S phase (in b). In contrast, transfection of full-length SALL1 into MCF-7 and E0771 breast cancer cells significantly induced tumor cell senescence (around 40%) and promoted cell cycle arrest in S phase. Breast cancer cells were transfected with the indicated constructs and cultured for additional 72 h. Senescent cells were analyzed using the SA-β-Gal activity assay and the cell cycle distribution in tumor cells was analyzed after incubation with propidium iodide. Data shown in (a) are mean ± SD from three independent experiments with similar results. **p < 0.01 compared with the mSALL1 and vector control groups. c and d Transfection of SALL1-S2E into MCF-7 and E0771 breast cancer cells lost the ability to induce tumor cell senescence. However, transfection of SALL1-S2A into breast cancer cells significantly augmented senescence induction in both cell lines compared with that of in wild type SALL1-transfected tumor cells. Cell transfection procedure and SA-β-Gal+ cell determination were identical to (a). SALL1-S2E : substitution of the serine with a glutamic acid in SALL1. SALL1-S2A : mutating the serine to an alanine in SALL1. SA-β-Gal+ tumor cells were identified with dark blue granules as indicated by the arrows (in c). Data shown in (d) are mean ± SD from three independent experiments with similar results. **p < 0.01, compared with the vector control group. #p < 0.01, compared with the wild type SALL1 group. e Transfection of wild type SALL1 and SALL1-S2A into MCF-7 tumor cells recruited NuRD complex components determined with GST pulldown analyses. In contrast, transfection of SALL1-S2E markedly disrupted recruitment of NuRD components. MCF-7 cells were transfected with or without plasmids pEBG-SALL1, pEBG-SALL1-S2A, and pEBG-SALL1-S2E, and cultured for 3 days. Total protein lysates precipitated with Protein G-Sepharose beads. Pulldowns were analyzed by western blotting with antibodies against SALL1, HDAC1, MTA2, MBD3 and RbAp46/48

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