The expression of DUSP1 gene is induced in human skin fibroblasts by oxidative/heat stress and growth factors. It specifies a protein with structural features similar to members of the non-receptor-type protein-tyrosine phosphatase family, and which has significant amino-acid sequence similarity to a Tyr/Ser-protein phosphatase encoded by the late gene H1 of vaccinia virus. The bacterially expressed and purified DUSP1 protein has intrinsic phosphatase activity, and specifically inactivates mitogen-activated protein (MAP) kinase in vitro by the concomitant dephosphorylation of both its phosphothreonine and phosphotyrosine residues. Furthermore, it suppresses the activation of MAP kinase by oncogenic ras in extracts of Xenopus oocytes. Thus, DUSP1 may play an important role in the human cellular response to environmental stress as well as in the negative regulation of cellular proliferation.
Background References
1. Szelenyi ER & Urso ML Time-course analysis of injured skeletal muscle suggests a critical involvement of ERK1/2 signaling in the acute inflammatory response. Muscle Nerve 45:552-61 (2012).
2. Asrih M et al. Role of ERK1/2 activation in microtubule stabilization and glucose transport in cardiomyocytes. Am J Physiol Endocrinol Metab 301:E836-43 (2011).
Sequence Similarity
Belongs to the protein-tyrosine phosphatase family. Non-receptor class dual specificity subfamily.
Tissue Specificity
Expressed at high levels in the lung, liver placenta and pancreas. Moderate levels seen in the heart and skeletal muscle. Lower levels found in the brain and kidney.
Post-translational Modification
Phosphorylation at Ser-359 and Ser-364 by MAPK1/ERK2 and MAPK3/ERK1 reduces its rate of degradation.
Western blot analysis of DUSP1 on different lysates with Rabbit anti-DUSP1 antibody (ET1701-82) at 1/2,000 dilution.
Lane 1: Jurkat cell lysate Lane 2: SK-Br-3 cell lysate Lane 3: NIH/3T3 cell lysate Lane 4: Mouse liver tissue lysate Lane 5: Mouse kidney tissue lysate Lane 6: Rat liver tissue lysate Lane 7: Rat kidney tissue lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 39 kDa Observed band size: 50 kDa
Exposure time: 5 minutes 10 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1701-82) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunocytochemistry analysis of NIH/3T3 cells labeling DUSP1 with Rabbit anti-DUSP1 antibody (ET1701-82) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-DUSP1 antibody (ET1701-82) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Immunocytochemistry analysis of C6 cells labeling DUSP1 with Rabbit anti-DUSP1 antibody (ET1701-82) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-DUSP1 antibody (ET1701-82) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Immunohistochemical analysis of paraffin-embedded human lung cancer tissue with Rabbit anti-DUSP1 antibody (ET1701-82) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1701-82) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse lung tissue with Rabbit anti-DUSP1 antibody (ET1701-82) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1701-82) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded rat lung tissue with Rabbit anti-DUSP1 antibody (ET1701-82) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1701-82) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
DUSP1 was immunoprecipitated from 0.2 mg SK-Br-3 cell lysate with ET1701-82 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1701-82 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.
Lane 1: SK-Br-3 cell lysate (input) Lane 2: ET1701-82 IP in SK-Br-3 cell lysate Lane 3: Rabbit IgG instead of ET1701-82 in SK-Br-3 cell lysate