Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC]

• ICC

Supplier Data

Immunocytochemistry - Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] (AB277803)

Immunofluorescence was performed on fixed and permeabilized Jurkat cells for detection of endogenous IRF9 using Anti-IRF9 Recombinant Rabbit Polyclonal Antibody (ab277803, 2 μg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal^™ Secondary Antibody, Alexa Fluor^® 488 conjugate (1/2000). Panel a) shows representative cells that were stained for detection and localization of IRF9 protein (green), Panel b) is stained for nuclei (blue) using SlowFade^® Gold Antifade Mountant with DAPI. Panel c) represents cytoskeletal F-actin staining using Alexa Fluor^® 555 Rhodamine Phalloidin (1/300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of IRF9. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

• WB

Supplier Data

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] (AB277803)

Western blot analysis was performed on whole cell extracts (30 μg lysate) of Jurkat (human T cell leukemia cell line from peripheral blood) (Lane 1), Jurkat treated with IFN-alpha (10 ng/mL for 16hr) (Lane 2), MCF7 (human breast adenocarcinoma cell line) (Lane 3), MCF7 treated with IFN-alpha (10 ng/mL for 16hr) (Lane 4), PC3 (human prostate adenocarcinoma cell line) (Lane 5) and PC3 treated with IFN-alpha (10 ng/mL for 16hr) (Lane 6).

The blots were probed with ab277803 at 1-2 μg/mL and detected by chemiluminescence using a Goat anti-Rabbit IgG (H+L)-HRP secondary antibody, (0.4 μg/mL, 1/2500 dilution).

4-12% Bis-Tris gel.

The membrane was probed with the relevant primary and secondary antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed.

All lanes:

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] (ab277803)

Predicted band size: 44 kDa

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• WB

Lab

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] (AB277803)

False colour image of Western blot : Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] staining at 2 μg/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab277803 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). 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 [14HCLC] (ab277803) at 2 µg/mL

Lane 1:

Wild-type A549 treated IFNg (10 IU/ml, 24 h) cell lysate at 20 µg

Lane 2:

IRF9 knockout A549 treated IFNg (10 IU/ml, 24 h) cell lysate at 20 µg

Lane 3:

Wild-type A549 vehicle control IFNg (0 IU/ml, 24 h) cell lysate at 20 µg

Lane 4:

IRF9 knockout A549 vehicle control IFNg (0 IU/ml, 24 h) 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 [14HCLC] (AB277803)

False colour image of Western blot : Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] staining at 2 μg/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab277803 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 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 [14HCLC] (ab277803) at 1/1000 dilution

Lane 1:

Wild-type HeLa treated 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 vehicle control 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

Predicted band size: 44 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] (AB277803)

False colour image of Western blot : Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] staining at 2 μg/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab277803 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 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 [14HCLC] (ab277803) at 2 µg/mL

Lane 1:

Wild-type HeLa treated 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 vehicle control 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

Predicted band size: 44 kDa

Observed band size: 48 kDa

false