Probable tumor suppressor initially identified as a CDK5R1 interactor controlling cell proliferation . Negatively regulates NF-kappa-B-mediated gene transcription through the control of RELA phosphorylation . Also regulates mitotic G2/M transition checkpoint and mitotic G2 DNA damage checkpoint. Through its interaction with CDKN2A/ARF and MDM2 may induce MDM2-dependent p53/TP53 ubiquitination, stabilization and activation in the nucleus, thereby promoting G1 cell cycle arrest and inhibition of cell proliferation . May play a role in the unfolded protein response, mediating the ufmylation of multiple proteins in response to endoplasmic reticulum stress . May also play a role in the rupture of the nuclear envelope during apoptosis . May regulate MAPK14 activity by regulating its dephosphorylation by PPM1D/WIP1
Background References
1. Chen J.et.al.A novel gene IC53 stimulates ECV304 cell proliferation and is upregulated in failing heart.Biochem. Biophys. Res. Commun. 294:161-166(2002).
Sequence Similarity
Belongs to the CDK5RAP3 family.
Tissue Specificity
Ubiquitously expressed. Expressed in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Isoform 3 is expressed in kidney, liver, skeletal muscle and placenta.
Post-translational Modification
May be phosphorylated by CDK5.; Ubiquitinated. Probably triggers proteasomal degradation and is negatively regulated by UFL1.; May be ufmylated.; Cleaved by caspases early during apoptosis, the resulting peptides may play a role in rupture of the nuclear envelope.
Western blot analysis of CDK5RAP3 on different lysates with Rabbit anti-CDK5RAP3 antibody (ER1902-72) at 1/5,000 dilution.
Lane 1: HeLa cell lysate Lane 2: A431 cell lysate Lane 3: MCF7 cell lysate Lane 4: EL4 cell lysate Lane 5: PC-12 cell lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 57 kDa Observed band size: 60 kDa
Exposure time: 1 minute 39 seconds; ECL: K1801;
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 (ER1902-72) at 1/5,000 dilution was used in primary antibody dilution (K1803) 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 EL4 cells labeling CDK5RAP3 with Rabbit anti-CDK5RAP3 antibody (ER1902-72) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-CDK5RAP3 antibody (ER1902-72) 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 (HA601187, 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 PC-12 cells labeling CDK5RAP3 with Rabbit anti-CDK5RAP3 antibody (ER1902-72) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-CDK5RAP3 antibody (ER1902-72) 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 (HA601187, 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 mouse stomach tissue with Rabbit anti-CDK5RAP3 antibody (ER1902-72) at 1/200 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 (ER1902-72) at 1/200 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 stomach tissue with Rabbit anti-CDK5RAP3 antibody (ER1902-72) at 1/200 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 (ER1902-72) at 1/200 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.
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