Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: IRAK-1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: Hek293 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab180747 observed at 85 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab180747 was shown to specifically react with IRAK-1 when IRAK-1 knockout samples were used. Wild-type and IRAK-1 knockout samples were subjected to SDS-PAGE. Ab180747 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with 800CW Goat anti-Rabbit and 680CW Goat anti-Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-IRAK-1 antibody [EPR18630] (ab180747) at 1/1000 dilution

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 3 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 77 kDa
Observed band size: 77 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 10 seconds; Lane 2: 3 minutes; Lane 3: 30 seconds; Lane 4: 1 minute.

There are two splice variants for IRAK-1, IRAK-1b and IRAK-1c, respectively.  The product recognizes full length IRAK-1 (80kDa).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling IRAK-1 with ab180747 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody  at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab180747 at 1/250 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells labeling IRAK-1 with ab180747 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on K562 cells. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab180747 at 1/250 dilution, followed by followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

IRAK-1 was immunoprecipitated from 0.35 mg of HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate with ab180747 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab180747 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: HEK-293 whole cell lysate 10µg (Input).

Lane 2: ab180747 IP in HEK-293 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab180747 in HEK-293 whole cell lysate.

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

Exposure time: 30 seconds.

The 68kDa band observed is likely a splice variant, according to PMID: 16690127.