Anti-SUZ12 antibody

• WB

Unknown

Western blot - Anti-SUZ12 antibody (AB12073)

All lanes:

Western blot - Anti-SUZ12 antibody (ab12073) at 1 µg/mL

Lane 1:

SW480 (Human colon adenocarcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 20 µg

Lane 3:

Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 83 kDa

Observed band size: 30 kDa,53 kDa,60 kDa,75 kDa,95 kDa

true

Exposure time: 4min

• WB

Lab

Western blot - Anti-SUZ12 antibody (AB12073)

Lanes 1 - 4 : Merged signal (red and green). Green - ab12073 observed at 95 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab12073 was shown to react with SUZ12 in western blot. The band observed in the SUZ12 knockout cell line ab264983 (SUZ12 knockout cell lysate ab257721) lane below 95 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab12073 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-SUZ12 antibody (ab12073) at 1 µg/mL

Lane 1:

Wild-type Hela cell lysate at 20 µg

Lane 2:

SUZ12 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SUZ12 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-suz12-knockout-hela-cell-line-ab264983'>ab264983</a>)

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

SUZ12 knockout HAP1 cell lysate at 20 µg

Predicted band size: 83 kDa

Observed band size: 95 kDa

false

• WB

Lab

Western blot - Anti-SUZ12 antibody (AB12073)

Lanes 1- 2 : Merged signal (red and green). Green - ab12073 observed at 95 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab12073 was shown to react with SUZ12 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264983 (knockout cell lysate ab257721) lane below 95kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and SUZ12 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab12073 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 μg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SUZ12 antibody (ab12073) at 1 µg/mL

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SUZ12 knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-suz12-knockout-hela-cell-lysate-ab257721'>ab257721</a>) at 20 µg

Predicted band size: 83 kDa

Observed band size: 95 kDa

false

• WB

Lab

Western blot - Anti-SUZ12 antibody (AB12073)

Lanes 1- 2 : Merged signal (red and green). Green - ab12073 observed at 95 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab12073 was shown to react with SUZ12 in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab264983 (CRISPR/Cas9 edited cell lysate ab257721) lane below 95kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and SUZ12 CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab12073 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 μg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SUZ12 antibody (ab12073) at 1 µg/mL

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

SUZ12 CRISPR/Cas9 edited HeLa cell lysate at 20 µg

Predicted band size: 83 kDa

Observed band size: 95 kDa

false

• WB

Lab

Western blot - Anti-SUZ12 antibody (AB12073)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : SUZ12 knockout HAP1 cell lysate (20 μg)
Lane 3 : Caco2 cell lysate (20 μg)
Lane 4 : MCF7 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab12073 observed at 95 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab12073 was shown to specifically recognize SUZ12 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when SUZ12 knockout samples were used. Wild-type and SUZ12 knockout samples were subjected to SDS-PAGE. ab12073 and ab8245 (loading control to GAPDH) were diluted to 1μg/mL and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-SUZ12 antibody (ab12073)

Predicted band size: 83 kDa

Observed band size: 95 kDa

false

• WB

Project

Western blot - Anti-SUZ12 antibody (AB12073)

All lanes:

Western blot - Anti-SUZ12 antibody (ab12073) at 1 µg/mL

Lane 1:

untransfected 293T cell lysate at 20 µg

Lane 2:

293T cells transfected with 5ug HA-Suz12 at 20 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG H&L (HRP) (Dako) at 1/2000 dilution

Predicted band size: 83 kDa

Observed band size: 76 kDa,95 kDa

false

• WB

CiteAb

Western blot - Anti-SUZ12 antibody (AB12073)

Western Blotting using Anti-SUZ12 antibody, ab12073. Publication image from Oliviero, S. et al., 2016, Nat Commun, 27301576. Legend direct from paper.

Myc represses Wnt pathway antagonists via PRC2.(a) In situ proximity ligation assay between Ezh2 or Eed and Myc, in LIF-ESCs, expressing either a control (shCtrl) or an Eed (shEed) shRNA. The proximity between Myc and an unrelated protein (IgG) was assessed as negative control. Scale bar, 50 µm. (b) Nuclear extracts obtained from ESCs were either immunostained (Input) or subjected to immunoprecipitation (IP) with indicated antibodies in the presence or absence of DNaseI. Immunostaining (IB) was performed using the indicated antibodies; 10% of the total protein samples were loaded as input. (c) Nuclear extracts from HEK 293 cell clones expressing the Flag-Myc or the Flag-MycδMBII were used to immunoprecipitate. Myc proteins with anti-Flag antibody and interacting proteins were revealed by immunostaining using the indicated antibodies. (d) Recombinant PRC2 complex was used to pull-down the His-Myc wt or the His-MycδMBII recombinant proteins. The PRC2 complex was bound to the Flag-resin and the Myc protein interactions were measured by immunostaining after peptide competition elution. (e) H3K27-histone methyltransferase activities (HMT) of the eluted protein complexes were measured by a colorimetric assay. (f) Schematic representation of the experiment : the control (shCtrl) and the double c- and N-Myc (dKD) ESCs were maintained, respectively, in LIF (black line) or in LIF+OHT (red line). At the indicated time point, the dKD ESCs were either grown in the presence of OHT (+OHT, red line) or shifted to LIF only medium (−OHT, blue line) for the following 16 h. Thereafter, the MycER protein was reactivated upon 4 h treatment with OHT (−/+ OHT, green line). (g,h) ChIP onMycER ESCs grown in the presence of LIF and expressing either a control (shCtrl) or Myc and Mycn (dKD) shRNAs. The levels of K4me3, K27me3, Myc, Suz12 and Ring1b at the TSS of the indicated genes was measured in the control cells (black bars), in the dKD cells maintained in the presence of OHT (red bars) or after OHT withdrawal for 16 h (blue bars), followed by the re-induction of MycER fusion protein by OHT treatment for additional 4 h (green bars). Data in panels g and h are means±s.d. (n=3). See also related Supplementary Figs 6 and 7.

false

• WB

CiteAb

Western blot - Anti-SUZ12 antibody (AB12073)

Western Blotting using Anti-SUZ12 antibody, ab12073. Publication image from Oliviero, S. et al., 2016, Nat Commun, 27301576. Legend direct from paper.

Myc represses Wnt pathway antagonists via PRC2.(a) In situ proximity ligation assay between Ezh2 or Eed and Myc, in LIF-ESCs, expressing either a control (shCtrl) or an Eed (shEed) shRNA. The proximity between Myc and an unrelated protein (IgG) was assessed as negative control. Scale bar, 50 µm. (b) Nuclear extracts obtained from ESCs were either immunostained (Input) or subjected to immunoprecipitation (IP) with indicated antibodies in the presence or absence of DNaseI. Immunostaining (IB) was performed using the indicated antibodies; 10% of the total protein samples were loaded as input. (c) Nuclear extracts from HEK 293 cell clones expressing the Flag-Myc or the Flag-MycδMBII were used to immunoprecipitate. Myc proteins with anti-Flag antibody and interacting proteins were revealed by immunostaining using the indicated antibodies. (d) Recombinant PRC2 complex was used to pull-down the His-Myc wt or the His-MycδMBII recombinant proteins. The PRC2 complex was bound to the Flag-resin and the Myc protein interactions were measured by immunostaining after peptide competition elution. (e) H3K27-histone methyltransferase activities (HMT) of the eluted protein complexes were measured by a colorimetric assay. (f) Schematic representation of the experiment : the control (shCtrl) and the double c- and N-Myc (dKD) ESCs were maintained, respectively, in LIF (black line) or in LIF+OHT (red line). At the indicated time point, the dKD ESCs were either grown in the presence of OHT (+OHT, red line) or shifted to LIF only medium (−OHT, blue line) for the following 16 h. Thereafter, the MycER protein was reactivated upon 4 h treatment with OHT (−/+ OHT, green line). (g,h) ChIP onMycER ESCs grown in the presence of LIF and expressing either a control (shCtrl) or Myc and Mycn (dKD) shRNAs. The levels of K4me3, K27me3, Myc, Suz12 and Ring1b at the TSS of the indicated genes was measured in the control cells (black bars), in the dKD cells maintained in the presence of OHT (red bars) or after OHT withdrawal for 16 h (blue bars), followed by the re-induction of MycER fusion protein by OHT treatment for additional 4 h (green bars). Data in panels g and h are means±s.d. (n=3). See also related Supplementary Figs 6 and 7.

false

• WB

CiteAb

Western blot - Anti-SUZ12 antibody (AB12073)

Western Blotting using Anti-SUZ12 antibody, ab12073. Publication image from Wong, K. K. et al., 2017, Nat Commun, 28387316. Legend direct from paper.

Loss of histone H3 lysine 27 trimethylation accompanies SCC transition.(a) Western blotting analysis performed on whole-cell extracts from tumours of the indicated genotypes and histologies. Histone H3 lysine 27 tri-methylation (H3K27me3) is markedly lower in SCC lesions; total histone H3 is the loading control. (b) Western blotting analysis performed on whole-cell extracts from tumours of the indicated genotypes and histologies. p63 and LKB1 confirm the histologies and genotypes of the lysates, and while EZH2 in more highly expressed in SCC tumours, the essential PRC2 subunit EED is absent in the SCC lesions. β-Actin is the loading control. (c) Immunohistochemisty for H3K27me3 on a panel of mouse lung tumours of the indicated histologies from KRAS/Lkb1, Pten/Lkb1/p53, and Pten/Lkb1 mice, scale bar, 50 µm. (d) Quantification of nuclear staining by dot analysis with Nikon software; data are mean±s.e.m. measured on serially stained sections, n=6–10. (e) Immunohistochemisty for H3K27me3 on a panel of human mixed adenocarcionomas, pure ADC and pure SCCs. Scale bar, 50 µm. (f) Quantification of nuclear staining, for H3K27me3 n=6 ADSCC, 14 ADC, 9 SCC, for EZH2 n=6 ADSCC, 7 ADC, 5 SCC, data are mean±s.e.m. measured on serially stained sections. P values represent 2 tailed t-test. See also Supplementary Fig. 3a–c.

false

• WB

CiteAb

Western blot - Anti-SUZ12 antibody (AB12073)

Western Blotting using Anti-SUZ12 antibody, ab12073. Publication image from Oliviero, S. et al., 2016, Nat Commun, 27301576. Legend direct from paper.

Myc represses Wnt pathway antagonists via PRC2.(a) In situ proximity ligation assay between Ezh2 or Eed and Myc, in LIF-ESCs, expressing either a control (shCtrl) or an Eed (shEed) shRNA. The proximity between Myc and an unrelated protein (IgG) was assessed as negative control. Scale bar, 50 µm. (b) Nuclear extracts obtained from ESCs were either immunostained (Input) or subjected to immunoprecipitation (IP) with indicated antibodies in the presence or absence of DNaseI. Immunostaining (IB) was performed using the indicated antibodies; 10% of the total protein samples were loaded as input. (c) Nuclear extracts from HEK 293 cell clones expressing the Flag-Myc or the Flag-MycδMBII were used to immunoprecipitate. Myc proteins with anti-Flag antibody and interacting proteins were revealed by immunostaining using the indicated antibodies. (d) Recombinant PRC2 complex was used to pull-down the His-Myc wt or the His-MycδMBII recombinant proteins. The PRC2 complex was bound to the Flag-resin and the Myc protein interactions were measured by immunostaining after peptide competition elution. (e) H3K27-histone methyltransferase activities (HMT) of the eluted protein complexes were measured by a colorimetric assay. (f) Schematic representation of the experiment : the control (shCtrl) and the double c- and N-Myc (dKD) ESCs were maintained, respectively, in LIF (black line) or in LIF+OHT (red line). At the indicated time point, the dKD ESCs were either grown in the presence of OHT (+OHT, red line) or shifted to LIF only medium (−OHT, blue line) for the following 16 h. Thereafter, the MycER protein was reactivated upon 4 h treatment with OHT (−/+ OHT, green line). (g,h) ChIP onMycER ESCs grown in the presence of LIF and expressing either a control (shCtrl) or Myc and Mycn (dKD) shRNAs. The levels of K4me3, K27me3, Myc, Suz12 and Ring1b at the TSS of the indicated genes was measured in the control cells (black bars), in the dKD cells maintained in the presence of OHT (red bars) or after OHT withdrawal for 16 h (blue bars), followed by the re-induction of MycER fusion protein by OHT treatment for additional 4 h (green bars). Data in panels g and h are means±s.d. (n=3). See also related Supplementary Figs 6 and 7.

false