All lanes : Anti-RIG-I/DDX58 antibody [EPR18629] (ab180675) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : DDX58 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 107 kDa
Observed band size: 107 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab180675 observed at 107 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab180675 was shown to react with DDX58 in A549 wild-type cells in western blot with loss of signal observed in DDX58 knockout cell line ab267117 (DDX58 knockout cell lysate ab257917). Wild-type and DDX58 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab180675 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-RIG-I/DDX58 antibody [EPR18629] (ab180675) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : DDX58 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 107 kDa
Observed band size: 107 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab180675 observed at 107 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab180675 was shown to react with DDX58 in wild-type A549 cells in western blot with loss of signal observed in DDX58 knockout cell line ab267116 (DDX58 knockout cell lysate ab257916). Wild-type and DDX58 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab180675 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: RIG-I/DDX58 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: K562 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab180675 observed at 107 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab180675 was shown to specifically react with RIG-I/DDX58 when RIG-I/DDX58 knockout samples were used. Wild-type and RIG-I/DDX58 knockout samples were subjected to SDS-PAGE. ab180675 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-RIG-I/DDX58 antibody [EPR18629] (ab180675) at 1/1000 dilution

Lane 1 : 293 (Human epithelial cells from embryonic kidney) whole cell lysate
Lane 2 : HeLa (Human epithelial cells 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 1/50000 dilution

Predicted band size: 107 kDa
Observed band size: 107 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-RIG-I/DDX58 antibody [EPR18629] (ab180675) at 1/1000 dilution + Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 107 kDa
Observed band size: 107 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-RIG-I/DDX58 antibody [EPR18629] (ab180675) at 1/1000 dilution + Human fetal kidney lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 107 kDa
Observed band size: 107 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-RIG-I/DDX58 antibody [EPR18629] (ab180675) at 1/1000 dilution + Human stomach lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 107 kDa
Observed band size: 107 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

RIG-I/DDX58 was immunoprecipitated from 1mg of Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate with ab180675 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab180675 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Jurkat whole cell lysate 10ug (Input). Lane 2: ab180675 IP in Jurkat whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab180675 in Jurkat whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.