Anti-DUSP4 antibody [EPR19881]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-DUSP4 antibody [EPR19881] (AB216576)

Flow cytometry overlay histogram showing left wild-type A549 positive cells and right negative DUSP4 knockout A549 stained with ab216576 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab216576) (1x 106 in 100μl at 1.0 μg/ml (1/1990)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (black line) was used at the same concentration and conditions as the primary antibody. Unlabelled sample was also used as a control (blue line).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-DUSP4 antibody [EPR19881] (AB216576)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MDA-MB-231 (Human breast adenocarcinoma cell line) cells labeling DUSP4 with ab216576 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on MDA-MB-231 cell line.

The nuclear counterstain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-DUSP4 antibody [EPR19881] (AB216576)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed MDA-MB-231 (Human breast adenocarcinoma cell line) cells labeling DUSP4 with ab216576 at 1/60 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-DUSP4 antibody [EPR19881] (AB216576)

ab216576 staining DUSP4 in wild-type A549 cells, with negative expression in DUSP4 knockout A549 cells. The cells were fixed with 100% methanol (5 min), permeabilised 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 overnight at +4°C with ab216576 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-DUSP4 antibody [EPR19881] (AB216576)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (Human lung carcinoma cell line) cells labeling DUSP4 with ab216576 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on A549 cell line.

The nuclear counterstain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• WB

Lab

Western blot - Anti-DUSP4 antibody [EPR19881] (AB216576)

Western blot : Anti-DUSP4 antibody [EPR19881] (ab216576) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab216576 was shown to bind specifically to DUSP4. A band was observed at 40 kDa in wild-type A549 cell lysates with no signal observed at this size in DUSP4 knockout cell line. To generate this image, wild-type and DUSP4 knockout 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 °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-DUSP4 antibody [EPR19881] (ab216576) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lanes 2 - 3:

DUSP4 knockout A549 cell lysate at 20 µg

Lane 4:

DUSP4 knockout A549 H15 cell lysate at 20 µg

Lane 5:

HCT 116 cell lysate at 20 µg

Lane 6:

MOLT-4 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 40 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-DUSP4 antibody [EPR19881] (AB216576)

DUSP4 was immunoprecipitated from 0.35mg of MDA-MB-231 (Human breast adenocarcinoma cell line) whole cell lysate with ab216576 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab216576 at 1/500 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1,000 dilution

Lane 1 : MDA-MB-231 whole cell lysate, 10μg (Input).

Lane 2 : ab216576 IP in MDA-MB-231 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab216576 in MDA-MB-231 whole cell lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-DUSP4 antibody [EPR19881] (ab216576)

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

• WB

Supplier Data

Western blot - Anti-DUSP4 antibody [EPR19881] (AB216576)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2 : 3 minutes; Lane 3 : 30 seconds; Lane 4 : 1 second.

All lanes:

Western blot - Anti-DUSP4 antibody [EPR19881] (ab216576) at 1/1000 dilution

Lane 1:

MDA-MB-231 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 2:

A549 (Human lung carcinoma cell line) whole cell lysate at 10 µg

Lane 3:

SK-BR-3 (Human mammary gland adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 4:

HCT 116 (Human colorectal carcinoma cell line) whole cell lysate at 10 µg

Secondary

Lanes 1 - 2:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Lanes 3 - 4:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

• WB

Supplier Data

Western blot - Anti-DUSP4 antibody [EPR19881] (AB216576)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 3 minutes; Lane 2/3 : 15 seconds.

All lanes:

Western blot - Anti-DUSP4 antibody [EPR19881] (ab216576) at 1/1000 dilution

Lane 1:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 3:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

• WB

Supplier Data

Western blot - Anti-DUSP4 antibody [EPR19881] (AB216576)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-DUSP4 antibody [EPR19881] (ab216576) at 1/1000 dilution

All lanes:

Human breast cancer lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-DUSP4 antibody [EPR19881] (AB216576)

Lanes 1 - 4 : Merged signal (red and green). Green - ab216576 observed at 40 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab216576 was shown to react with DUSP4 in wild-type A549 cells in Western blot with loss of signal observed in DUSP4 knockout cell line ab273859 (DUSP4 knockout cell lysate ab273813). Wild-type A549 and DUSP4 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab216576 and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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 h at room temperature before imaging.

All lanes:

Western blot - Anti-DUSP4 antibody [EPR19881] (ab216576) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

DUSP4 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human DUSP4 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-dusp4-knockout-a549-cell-line-ab273859'>ab273859</a>)

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

MOLT-4 cell lysate at 20 µg

Predicted band size: 43 kDa

Observed band size: 40 kDa

false