All lanes : Anti-DUSP6 antibody [EPR129Y] (ab76310) at 1/4000 dilutionLane 1 : MarkerLane 2 : Lysate from wild type primary murine embryonic fibroblasts (MEFs) untreated at 10 µgLane 3 : Lysate from wild type primary murine embryonic fibroblasts (MEFs) treated with 100ngµl-1, 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 hours at 10 µgLane 4 : Lysate from MKP-3 null primary murine embryonic fibroblasts (MEFs) untreated at 10 µgLane 5 : Lysate from MKP-3 null primary murine embryonic fibroblasts (MEFs) treated with 100ngµl-1, 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 hours at 10 µgSecondaryGoat anti Rabbit HRP conjugate at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.

Anti-DUSP6 antibody [EPR129Y] (ab76310) at 1/500 dilution + 3T3 cell lysate at 10 µgSecondaryHRP labelled goat anti-rabbit at 1/2000 dilution

Immunohistochemical staining of paraffin-embedded human gastric carcinoma using ab76310 at 1/50 dilution.

ICC/IF image of ab76310 stained PC12 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab76310, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Overlay histogram showing HeLa cells stained with ab76310 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76310, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized with 0.1% PBS-Tween 20 used under the same conditions.