Cell cycle events are regulated by the sequential activation and deactivation of cyclin dependent kinases (Cdks) and by the proteolysis of cyclins. The cell division control (Cdc) genes are required at various points in the cell cycle. Cdc40, also known as pre-mRNA-processing factor 17 (PRPF17) or EH-binding protein 3 (EHB3), is a 579 amino acid nuclear protein. Cdc40 is essential for the catalytic step II of the pre-mRNA splicing process, in which Cdc40 associates with the spliceosome C complex. Cdc40 contains seven WD repeats, which are important in protein-protein interactions. Cdc40 has sequence similarity to the yeast protein Prp17, which is involved in pre-mRNA splicing and cell cycle progression. The sequence similarity between the mammalian Cdc40 and the yeast Prp17 may indicate an additional role in cell cycle progression for mammalian Cdc40.
Background References
1. Olsen JV, et al. 2006. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell. 127 (3): 635–48.
2. Barrios-Rodiles M, et al. 2005. High-throughput mapping of a dynamic signaling network in mammalian cells. Science. 307 (5715): 1621–5.
Western blot analysis of CDC40 on different lysates with Rabbit anti-CDC40 antibody (ET1612-85) at 1/5,000 dilution.
Lane 1: K-562 cell lysate (20 µg/Lane) Lane 2: HCT 116 cell lysate (20 µg/Lane) Lane 3: Neuro-2a cell lysate (20 µg/Lane) Lane 4: Mouse lung tissue lysate (40 µg/Lane) Lane 5: Rat brain tissue lysate (40 µg/Lane)
Predicted band size: 66 kDa Observed band size: 66 kDa
Exposure time: 18 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 (ET1612-85) 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 Neuro-2a cells labeling CDC40 with Rabbit anti-CDC40 antibody (ET1612-85) 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-CDC40 antibody (ET1612-85) 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 C6 cells labeling CDC40 with Rabbit anti-CDC40 antibody (ET1612-85) 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-CDC40 antibody (ET1612-85) 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 rat brain tissue using anti-CDC40 antibody. Counter stained with hematoxylin.
Immunohistochemical analysis of paraffin-embedded mouse colon tissue using anti-CDC40 antibody. Counter stained with hematoxylin.
Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-CDC40 antibody. Counter stained with hematoxylin.
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