Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line

• sELISA

Lab

Sandwich ELISA - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (AB273379)

Human IL-1RA concentration was interpolated from the IL-1RA standard curve. Supernatants from cell culture samples were serially diluted and assessed by the Human IL-1ra ELISA Kit (ab211650). Wild-type A-431, IL1RN knockout A-431 (ab273379) and THP-1 cells were assessed in duplicate (n=2). Data are represented as the mean and error bars represent standard deviation.

• WB

Lab

Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (AB273379)

False colour image of Western blot : Anti-IL-1RA antibody [EPR6483] staining at 1/50000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab124962 was shown to bind specifically to IL-1RA. A band was observed at 18 kDa in wild-type A431 cell lysates with no signal observed at this size in IL1RN knockout cell line ab273379 (knockout cell lysate ab275530). To generate this image, wild-type and IL1RN knockout A431 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-IL-1RA antibody [EPR6483] (<a href='/ko/products/primary-antibodies/il-1ra-antibody-epr6483-ab124962'>ab124962</a>) at 1/50000 dilution

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell lysate (<a href='/ko/products/cell-lysates/human-il1rn-il1-receptor-antagonist-knockout-a-431-cell-lysate-ab275530'>ab275530</a>) at 20 µg

Lane 3:

MOLT-4 cell lysate at 20 µg

Lane 4:

Human Kidney cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

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

Observed band size: 18 kDa

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• WB

Lab

Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (AB273379)

False colour image of Western blot : Anti-IL-1RA antibody [EPR6483] staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab124962 was shown to bind specifically to IL-1RA. A band was observed at 19 kDa in wild-type A431 cell lysates with no signal observed at this size in IL1RN knockout cell line ab273379 (knockout cell lysate ab275530). To generate this image, wild-type and IL1RN knockout A431 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-IL-1RA antibody [EPR6483] (<a href='/ko/products/primary-antibodies/il-1ra-antibody-epr6483-ab124962'>ab124962</a>) at 1/10000 dilution

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

IL-1RA knockout A431 cell lysate at 20 µg

Lane 2:

Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)

Predicted band size: 20 kDa

Observed band size: 19 kDa

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• ICC

Lab

Immunocytochemistry - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (AB273379)

IL-1RA staining observed in wild-type A431 cells (top panel) and IL1RN knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 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 an anti-IL-1ra antibody at 10μg/ml concentration (shown in green) and ab195889 (Mouse monoclonal to alpha Tubulin - Alexa Fluor^® 594) at 1/250 dilution (shown in red) overnight at 4°C. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• WB

Lab

Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (AB273379)

Lanes 1 - 4 : Merged signal (red and green). Green - Anti-IL-1ra antibody observed at 18 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
Anti-IL-1ra antibody was shown to react with IL-1ra in wild-type A431 cells in Western blot with loss of signal observed in IL-1RA knockout cell line ab273379 (IL-1RA knockout cell lysate ab275530). Wild-type A431 and IL-1RA knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with anti-IL-1ra antibody and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 ^°C at 0.5 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Donkey anti-Goat IgG H&L (IRDye^® 800CW) preabsorbed (ab216775) and Donkey anti-Mouse 680RD secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Anti-IL-1ra antibody at 0.5 µg/mL

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

IL-1RA knockout A431 cell lysate at 20 µg

Lane 2:

Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

SK-OV-3 cell lysate at 20 µg

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• Sanger seq

Unknown

Sanger Sequencing - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (AB273379)

49 bp deletion in exon 5