Anti-DDIT3 antibody [9C8] (ab11419)

False colour image of Western blot: Anti-DDIT3 antibody [9C8] staining at 5µg/ml, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab11419 was shown to bind specifically to DDIT3. A band was observed at 25 kDa in wild-type y cell lysates with no signal observed at this size in DDIT3 knockout cell line ab265760 (knockout cell lysate ab256889). To generate this image, wild-type and DDIT3 knockout y cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

Lanes 1 - 5: Merged signal (red and green). Green - ab11419 observed at 26 kDa. Red - loading control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.

ab11419 was shown to react with DDIT3 in wild-type SW480 cells in western blot with loss of signal observed in DDIT3 knockout sample. Wild-type and DDIT3 knockout SW480 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab11419 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at 5 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab11419 staining DDIT3 in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells +/- Tunicamycin 1.5μM, 6 hours (ab120296). 

The cells were fixed with 4% PFA (10 min), permeabilized with 0.1% Triton-X for 5 mins 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 overnight at +4°C with ab11419 at 5μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Lanes 1 - 5: Merged signal (red and green). Green - ab11419 observed at 27 kDa. Red - loading control, Rabbit anti Actin observed at 42kDa.

ab11419 was shown to react with DDIT3 in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab11419 and Rabbit anti Actin overnight at 4°C at 5 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Treated with 20µg/ml poly(I:C).

See Abreview

See Abreview

ab11419 staining DDIT3 in SK-N-SH (human neuroblastoma cell line) cells treated with deltamethrin (ab141019), by ICC/IF. Increase of DDIT3 expression correlates with increased concentration of deltamethrin, as described in literature.
The cells were incubated at 37°C for 48 hours in media containing different concentrations of ab141019 (deltamethrin) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab11419 (10 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight^® 488 anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.