Carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD) is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. This trifunctional protein which is associated with the enzymatic activities of the first 3 enzymes in the 6-step pathway of pyrimidine biosynthesis is the rate-limiting step in the de novo pyrimidine synthetic pathway. Although most of the CAD protein in the cell is cytosolic, phosphorylation at threonine 456 localizes the protein to the nucleus. While MAPK and EGF phosphorylate CAD at threonine 456, MAPK and c-myc have been found to induce over-expression of CAD.
ICC/IF image of ab97340 stained HepG2 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 (ab97340, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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.