The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. This gene encodes a trifunctional protein which is associated with the enzymatic activities of the first 3 enzymes in the 6-step pathway of pyrimidine biosynthesis: carbamoylphosphate synthetase (CPS II), aspartate transcarbamoylase, and dihydroorotase. This protein is regulated by the mitogen-activated protein kinase (MAPK) cascade, which indicates a direct link between activation of the MAPK cascade and de novo biosynthesis of pyrimidine nucleotides. Alternative splicing results in multiple transcript variants encoding different isoforms.

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Immunocytochemical staining of HeLa cells using CAD antibody , Top panel: wild-type (WT); Bottom panal: CAD shRNA knockdown (KD). Nuclei were stained blue with DAPI; Acyl-CoA dehydrogenase short chain was stained magenta with Alexa Fluor® 647. Scale bar, 20 μm.

Flow cytometric analysis of CAD expression in HepG2 cells using CAD antibody . Green, isotype control; red, CAD.

Immunocytochemical staining of HepG2 cells with CAD antibody . Nuclei were stained blue with DAPI; CAD was stained magenta with Alexa Fluor® 647. Images were taken using Leica stellaris 5. Protein abundance based on laser Intensity and smart gain: Medium. Scale bar, 20 μm.

Western blotting analysis using CAD antibody . Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with CAD antibody and HRP-conjugated goat anti-rabbit secondary antibody respectively. Image was developed using NaQ™ ECL Substrate Kit .

Western blotting analysis using CAD antibody . CAD expression in wild-type (WT) and CAD shRNA knockdown (KD) HeLa cells with 20 μg of total cell lysates. Hsp90 α serves as a loading control. The blot was incubated with CAD antibody and HRP-conjugated goat anti-rabbit secondary antibody respectively. Image was developed using FeQ™ ECL Substrate Kit .

Validation of CAD knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HeLa cells were stained with CAD antibody and analyzed using BD flow cytometer.

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