Anti-HOXA9 antibody [EPR3655(2)]

• IP

Supplier Data

Immunoprecipitation - Anti-HOXA9 antibody [EPR3655(2)] (AB140631)

Lane 1 : Wild-type HEK-293 input Lane 2 : HOXA9 knockout HEK-293 input Lane 3 : Wild-type HEK-293 immunoprecipitated with anti-HOXA9 antibody ab140631 Lane 4 : HOXA9 knockout HEK-293 immunoprecipitated with anti-HOXA9 antibody ab140631 Lane 5 : Wild-type HEK-293 immunoprecipitated with isotype control antibody ab172730 Lane 6 : HOXA9 knockout HEK-293 immunoprecipitated with isotype control antibody ab172730 HOXA9 was immunoprecipitated with Recombinant Anti-HOXA9 antibody [EPR3655(2)] (ab140631) at 10 µg ml-1. A non-specific band detected in the input material at 37 kDa was not immunoprecipitated. ab172730 was used as the isotype control. Western blot was performed on immunoprecipitates of wild-type and HOXA9 knockout HEK-293 cell lysates (ab273706). To generate this image, wild-type and HOXA9 knockout HEK-293 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 VeriBlot for IP Detection Reagent (HRP) (ab131366) for 1 h at room temperature, washed again four times then imaged.

All lanes:

Immunoprecipitation - Anti-HOXA9 antibody [EPR3655(2)] (ab140631) at 10 µg/mL

Lane 1:

Wild-type HEK-293 input

Lane 2:

HOXA9 knockout HEK-293 input

Lane 3:

Wild-type HEK-293 immunoprecipitated with anti-HOXA9 antibody ab140631

Lane 4:

HOXA9 knockout HEK-293 immunoprecipitated with anti-HOXA9 antibody ab140631

Lane 5:

Wild-type HEK-293 immunoprecipitated with isotype control antibody <a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>

Lane 6:

HOXA9 knockout HEK-293 immunoprecipitated with isotype control antibody <a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>)

Predicted band size: 30 kDa

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• WB

Lab

Western blot - Anti-HOXA9 antibody [EPR3655(2)] (AB140631)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-HOXA9 antibody [EPR3655(2)] (ab140631) at 1/2000 dilution

Lane 1:

HepG2 whole cell lysate at 20 µg

Lane 2:

Human thymus lysate at 20 µg

Lane 3:

U937 whole cell lysate at 20 µg

Lane 4:

HEK293 whole cell lysate at 20 µg

Secondary

All lanes:

Anti-rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 30 kDa

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• WB

Lab

Western blot - Anti-HOXA9 antibody [EPR3655(2)] (AB140631)

False colour image of Western blot : Anti-HOXA9 antibody [EPR3655(2)] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab140631 was shown to bind specifically to HOXA9. A band was observed at 35 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in HOXA9 knockout cell line. The HOXA9 band is at approximately 35 kDa. This band is present in the nuclear fraction and is absent in the KO. The identity of the brighter band at 37 kDa is unknown but is non-specific as it is present in all samples. To generate this image, wild-type and HOXA9 knockout HEK-293 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-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-HOXA9 antibody [EPR3655(2)] (ab140631) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 Nuclear Fremont cell lysate at 20 µg

Lane 2:

Wild-type HEK-293 Cytoplasmic Fremont cell lysate at 20 µg

Lane 3:

HOXA9 knockout HEK-293 Nuclear Fremont cell lysate at 20 µg

Lane 4:

HOXA9 knockout HEK-293 Cytoplasmic Fremont cell lysate at 20 µg

Lane 5:

Wild-type HEK-293 Nuclear Oxford Genetics cell lysate at 20 µg

Lane 6:

Wild-type HEK-293 Cytoplasmic Oxford Genetics cell lysate at 20 µg

Lane 7:

HOXA9 knockout HEK-293 Nuclear Oxford Genetics cell lysate at 20 µg

Lane 8:

HOXA9 knockout HEK-293 Cytoplasmic Oxford Genetics cell lysate at 20 µg

Lane 9:

HEK-293 Non-transfected control Nuclear cell lysate at 20 µg

Lane 10:

HEK-293 Non-transfected control Cytoplasmic cell lysate at 20 µg

Lane 11:

HEK-293 scrambled siRNA transfected Nuclear cell lysate at 20 µg

Lane 12:

HEK-293 scrambled siRNA transfected Cytoplasmic cell lysate at 20 µg

Lane 13:

HEK-293 HOXA9 siRNA transfected Nuclear cell lysate at 20 µg

Lane 14:

HEK-293 HOXA9 siRNA transfected Cytoplasmic cell lysate at 20 µg

Predicted band size: 30 kDa

Observed band size: 35 kDa

true

• WB

Unknown

Western blot - Anti-HOXA9 antibody [EPR3655(2)] (AB140631)

All lanes:

Western blot - Anti-HOXA9 antibody [EPR3655(2)] (ab140631) at 1/1000 dilution

Lane 1:

HepG2 cell lysate at 10 µg

Lane 2:

Human thymus tissue lysate at 10 µg

Lane 3:

U937 cell lysate at 10 µg

Lane 4:

SW480 ell lysate at 10 µg

Lane 5:

293T cell lysate at 10 µg

Predicted band size: 30 kDa

false

• WB

Lab

Western blot - Anti-HOXA9 antibody [EPR3655(2)] (AB140631)

Western blot : Anti-HOXA9 antibody [EPR3655(2)] (ab140631) staining at 1/1000 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab140631 was shown to bind specifically to HOXA9. A band was observed at 30 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in HOXA9 knockout cell line. To generate this image, wild-type and HOXA9 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 before development with a high-sensitivity ECL substrate kit and imaged with 1 minute exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-HOXA9 antibody [EPR3655(2)] (ab140631) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human HOXA9 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-hoxa9-knockout-hct116-cell-line-ab287488'>ab287488</a>)

Lane 2:

HOXA9 knockout HCT 116 cell lysate at 20 µg

Secondary

Lanes 1 - 2:

HRP conjugated Goat anti-Rabbit (H+L) at 1/20000 dilution

Lanes 1 - 2:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 30 kDa

false

• WB

CiteAb

Western blot - Anti-HOXA9 antibody [EPR3655(2)] (AB140631)

Western Blotting using Anti-HOXA9 antibody [EPR3655(2)], ab140631. Publication image from Nguyen, D. T. T. et al., 2020, Nat Commun, 32332729. Legend direct from paper.

Differential control of MSI2 targets in LSCs compared with normal LSKs.Diff.frequency at various sites identified by MSI2-HyperTRIBE in Hoxa9 3′UTR (a), Ikzf2 3′UTR (b) and Myb 3′UTR (c) in LSKs and LSCs. Numbers on the X-axis is the start and end of 3′UTR. Data presented as the mean values from three independent HyperTRIBE experiments. Significant difference is determined by beta-binomial test. * adjusted p < 0.1 d Representative immunoblot images and quantitation showing no significant change in HOXA9, IKZF2 and MYB protein expression upon Msi2 knockout in LSKs after 3 weeks of pIpC treatment in Msi2 f/f Cre(–) and Cre(+) mice. Each data point is an independent treated mouse. Data are presented as mean ± SEM. Two-sided unpaired Student t test. ***p < 0.005. (p = 0.002 for MSI2). e Representative Immunoblot images and quantitation showing significant decrease in HOXA9, IKZF2 and MYB protein expression upon Msi2 knockout at 68 h after TAM treatment in MLL-AF9 Msi2 CreER(+) LSCs. Each data point is an independent treated mouse. Data are presented as mean ± SEM. N = 6 independent experiments for HOXA9 and IKZF2, n = 3 independent experiments for MYB. Two-sided paired Student t test. **p < 0.01, ***p < 0.001; ****p < 0.0001. (p = 0.000002 for MSI2, p = 0.00015 for HOXA9, p = 0.019 for IKZF2, p = 0.000019 for MYB). f Schematic depiction of MSI2 elevated RNA binding and reduction of target protein expression upon MSI2 ablation in LSCs, but not in LSKs.

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