Human IRF9 (Interferon regulatory factor 9) knockout A549 cell line

• ICC

Lab

Immunocytochemistry - Human IRF9 (Interferon regulatory factor 9) knockout A549 cell line (AB267119)

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).

• WB

Lab

Western blot - Human IRF9 (Interferon regulatory factor 9) knockout A549 cell line (AB267119)

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°. 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] (<a href='/en-us/products/primary-antibodies/interferon-regulatory-factor-9-irf-9-antibody-epr24260-55-ab271043'>ab271043</a>) 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 (ab267119)

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

• Sanger seq

Unknown

Sanger Sequencing - Human IRF9 (Interferon regulatory factor 9) knockout A549 cell line (AB267119)

Allele-1 : 5 bp deletion in exon2

• Sanger seq

Unknown

Sanger Sequencing - Human IRF9 (Interferon regulatory factor 9) knockout A549 cell line (AB267119)

Allele-2 : 5 bp insertion in exon 2.