Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

Immunohistochemical analysis of paraffin-embedded HeLa (human cervix adenocarcinoma epithelial cell) cells (A) and HeLa cells treated by 10 ng/ml IFN for 18h (B) tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Almost no staining on HeLa cells (A) and strongly positive staining on HeLa cells treated by 10 ng/ml IFN for 18h (B). The section was incubated with ab271043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

ab271043 staining Interferon regulatory factor 9 in wild-type HeLa cells and IRF9 knockout HeLa cells treated with interferon-a1 (ab48750) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab271043 at 0.4 μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/50 dilution(9.3 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/500 dilution (Green). Confocal image showing increased nuclear and weak cytoplasmic staining in HeLa cells treated with Interferon alpha-1 (10 ng/ml) for 16 h is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/500 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

Immunohistochemical analysis of paraffin-embedded Clear cell carcinoma of human kidney tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on Clear cell carcinoma of human kidney (PMID : 30355451). The section was incubated with ab271043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use (Bond™ Polymer Refine Detection). Positive staining on human spleen. The section was incubated with ab271043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

Immunohistochemical analysis of paraffin-embedded Human lung tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human lung (PMID : 30355451). The section was incubated with ab271043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC

Lab

Immunocytochemistry - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

ab271043 staining Interferon regulatory factor 9 in wild-type A549 cells and IRF9 knockout A549 cells treated with interferon-a1 (ab48750) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab271043 at 0.4 μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• IP

Supplier Data

Immunoprecipitation - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

Interferon regulatory factor 9/IRF-9 was immunoprecipitated from 0.35 mg treated HeLa whole cell lysate with ab271043 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271043 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 10 ng/ml human IFN alpha 1 (ab48750) for 16 hours, whole cell lysate 10 ug

Lane 2 : ab271043 IP in HeLa treated with 10 ng/ml human IFN alpha 1 (ab48750) for 16 hours whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab271043 in HeLa treated with 10 ng/ml human IFN alpha 1 (ab48750) for 16 hours whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043)

Predicted band size: 44 kDa

Observed band size: 48 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

Interferon regulatory factor 9/IRF-9 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) whole cell lysate with ab271043 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271043 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : THP-1 whole cell lysate 10 ug

Lane 2 : ab271043 IP in THP-1 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab271043 in THP-1 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043)

Predicted band size: 44 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

False colour image of Western blot : Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab271043 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in IRF9 knockout cell line ab266051 (knockout cell lysate ab258472). The band observed in the knockout lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and IRF9 knockout HeLa cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution

Lane 1:

wild-type HeLa Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate at 20 µg

Lane 2:

IRF9 knockout HeLa treated: hIFN-a1 (10 ng/ml, 16 h) cell lysate at 20 µg

Lane 3:

wild-type HeLa Control Interferon-alpha1 (hIFN-a1) (0 ng/ml, 16 h) cell lysate at 20 µg

Lane 4:

IRF9 knockout HeLa vehicle control: hIFN-a1 (0 ng/ml, 16 h) cell lysate at 20 µg

Lane 5:

THP-1 cell lysate at 20 µg

Lane 6:

ACHN cell lysate at 20 µg

Predicted band size: 44 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

IFR9 can be degraded by the EV71-encoded 3C protease. A 48-kDa full length and 30-kDa cleaved IRF9 are observed. The molecular weight is consistent with what have been described in literature (PMID : 21536800).

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution

Lane 1:

Human ovary cancer tissue lysate at 40 µg

Lane 2:

Human liver cancer tissue lysate at 40 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 44 kDa

Observed band size: 30 kDa,48 kDa

false

• WB

Lab

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

False colour image of Western blot : Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab271043 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type A549 cell lysates with no signal observed at this size in IRF9 CRISPR-Cas9 edited cell line ab267119 (CRISPR-Cas9 edited cell lysate ab258473). The band observed in the CRISPR-Cas9 edited lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and IRF9 CRISPR-Cas9 edited A549 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-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution

Lane 1:

wild-type A549 Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate at 20 µg

Lane 2:

IRF9 knockout A549 Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate at 20 µg

Lane 2:

Western blot - Human IRF9 (Interferon regulatory factor 9) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-irf9-interferon-regulatory-factor-9-knockout-a549-cell-line-ab267119'>ab267119</a>)

Lane 3:

wild-type A549 Control Interferon-alpha1 (hIFN-a1) (0 ng/ml, 16 h) cell lysate at 20 µg

Lane 4:

IRF9 knockout A549 Control Interferon-alpha1 (hIFN-a1) (0 ng/ml, 16 h) cell lysate at 20 µg

Lane 5:

THP-1 cell lysate at 20 µg

Lane 6:

ACHN cell lysate at 20 µg

Predicted band size: 44 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

False colour image of Western blot : Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab271043 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in IRF9 CRISPR-Cas9 edited cell line ab266051 (CRISPR-Cas9 edited cell lysate ab258472). The band observed in the CRISPR-Cas9 edited lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and IRF9 CRISPR-Cas9 edited HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution

Lane 1:

wild-type HeLa Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate at 20 µg

Lane 2:

IRF9 knockout HeLa treated: hIFN-a1 (10 ng/ml, 16 h) cell lysate at 20 µg

Lane 2:

Western blot - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-irf9-interferon-regulatory-factor-9-knockout-hela-cell-line-ab266051'>ab266051</a>)

Lane 3:

wild-type HeLa Control Interferon-alpha1 (hIFN-a1) (0 ng/ml, 16 h) cell lysate at 20 µg

Lane 4:

IRF9 knockout HeLa vehicle control: hIFN-a1 (0 ng/ml, 16 h) cell lysate at 20 µg

Lane 5:

THP-1 cell lysate at 20 µg

Lane 6:

ACHN cell lysate at 20 µg

Predicted band size: 44 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

False colour image of Western blot : Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab271043 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type A549 cell lysates with no signal observed at this size in IRF9 knockout cell line ab267119 (knockout cell lysate ab258473). The band observed in the knockout lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and IRF9 knockout A549 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-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution

Lane 1:

wild-type A549 Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate at 20 µg

Lane 2:

IRF9 knockout A549 Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate at 20 µg

Lane 3:

wild-type A549 Control Interferon-alpha1 (hIFN-a1) (0 ng/ml, 16 h) cell lysate at 20 µg

Lane 6:

ACHN cell lysate at 20 µg

Predicted band size: 44 kDa

false

• WB

Lab

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (AB271043)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

IRF9 expression is increased by IFN-alpha (PMID : 18190617).

Exposure time : 15 seconds.

All lanes:

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

HeLa treated with 10ng/ml human IFN alpha 1 (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-alpha-1-protein-active-ab48750'>ab48750</a>) for 16 hours, whole cell lysate at 40 µg

Lane 3:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 40 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 44 kDa

Observed band size: 48 kDa

false