General description

Cullin-4A (UniProt Q13619; also known as CUL-4A) is encoded by the CUL4A gene (Gene ID 8451) in human. CUL-4A belongs to the cullin family of hydrophobic proteins that functions as the scaffold component of E3 ubiquitin ligase complexes. CUL-4A interacts with DNA damage binding protein 1 (DDB1) and ring of cullins (ROC1) to assemble the E3 complex, where DDB1 acts a linker protein for CUL4 and recruits substrates to the CUL4-ROC1 catalytic core through interaction with a subset of WD40 proteins. At least 90 DDB1-binding WD40 (DWD) proteins (also known as DCAF for Ddb1- and Cul4-associated factors and CDW for CUL4 and DDB1-associated WD40 repeats) are known, suggesting a large number of potential substrates for CUL4-ROC1 ligase complexes. CUL4-ROC1 E3 ligase complexes play important roles in cell cycle, DNA replication, genomic stability, haematopoiesis, and spermatogenesis. CUL-4A is highly expressed in testis and spleen, while poor expression is found in lung, liver, thymus, small intestine, and muscular tissues. CUL4A gene amplification has been demonstrated in many kinds of tumors, including hepatocellular carcinomas (HCC), squamous cell carcinomas, and adrenocortical carcinoma. CUL4A silencing is reported to suppress the motility of HCC cells and reverse their epithelial-mesenchymal transition (EMT) tendency.

Specificity

This polyclonal antibody was raised againt human Cullin-4A spliced isoform 1 N-terminal end sequence not present in spliced isoform 2. This polyclonal antibody detects both non-NEDDylated and NEDDylated Cullin-4A (McCall, C.M., et al. (2008). Mol. Cell. Biol. 28(18):5621-5633; Hu, J., et al. (2004). Nat. Cell Biol. 6(10):1003-1009).

Immunogen

Epitope: N-terminus

Synthetic linear peptide corresponding to human Cullin-4A spliced isoform 1 N-terminal end sequence.

Application

Detect Cul4A using this rabbit polyclonal Anti-Cullin-4A, Cat. No. ABS1584, validated for use in Immune Complex Enzyme Assay, Immunoprecipitation, and Western Blotting.

Research Category
Signaling

Western Blotting Analysis: A 1:500 dilution from a representative lot detected Cullin-4A in 10 µg of HeLa cell lysate.

Enzyme Assay: A representative lot immunoprecipitated cullin-4A complex from HeLa cell lysate for a subsequent in vitro Myc-CDT1 ubiquitination reaction in the presence of ubiquitin (Ub), E1 & E2 enzymes. RNAi-mediated DDB1, but not DDB2, knockdown prior to IP prevented a successful cullin-4A immune complex-dependent ubiquitination reaction (Hu, J., et al. (2004). Nat. Cell Biol. 6(10):1003-1009).

Immunoprecipitation Analysis: Representative lots immunoprecipitated both unmodified and NEDD8-modified (NEDDylated) endogenouse cullin-4A together with associated proteins (including CAND1, CSN1-8, DDB1, and VprBP) from human BT474, U2OS, HEK293T, and HeLa cells (McCall, C.M., et al. (2008). Mol. Cell. Biol. 28(18):5621-5633; Hu, J., et al. (2004). Nat. Cell Biol. 6(10):1003-1009).

Immunoprecipitation Analysis: A representative lot immunoprecipitated Myc3-cullin-4A HEK293T cells. Myc-CDT1 was co-immunoprecipitated with Myc3-cullin-4A only in the presence of exogenously expressed T7-DDB1. RNAi-mediated CAND1-knockdown enhanced T7-DDB1 co-IP, while DDB1 knockdown enhanced myc-CAND1 co-IP (Hu, J., et al. (2004). Nat. Cell Biol. 6(10):1003-1009).

Western Blotting Analysis: A representative lot detected both unmodified and NEDD8-modified (NEDDylated) endogenous cullin-4A in whole cell lysates (U2OS, WI-38/E6, HeLa) or chromatin fractions (HeLa and HCT116), in VprBP and cullin-4A immunoprecipitates (U2OS), as well as in VprBP-, DDB1-, CDT1-, CSN5-containing complexes fractionated from HeLa S3 cell lysate by gel filtration (McCall, C.M., et al. (2008). Mol. Cell. Biol. 28(18):5621-5633).

Western Blotting Analysis: A representative lot detected both unmodified and NEDD8-modified (NEDDylated) endogenous cullin-4A in HEK293T and HeLa cell lysates, as well as in T7-tag and Myc-tag immunoprecipitates from HEK293T cells expressing transfected T7-DDB1 and Myc-CAND1 (Hu, J., et al. (2004). Nat. Cell Biol. 6(10):1003-1009).

Quality

Evaluated by Western Blotting in Jurkat cell lysate.

Western Blotting Analysis: A 1:500 dilution of this antibody detected Cullin-4A in 10 µg of Jurkat cell lysate.

Target description

~80 kDa observed. ~82 kDa NEDDylated form (NEDD8 modified) was also observed upon longer exposure. 87.68 kDa calculated. Uncharacterized bands may be observed in some lysate(s).

Physical form

Affinity purified.

Purified rabbit polyclonal antibody in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide

Storage and Stability

Stable for 1 year at 2-8°C from date of receipt.

Other Notes

Concentration: Please refer to lot specific datasheet.

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

biological source: rabbit. Quality Level: 100. antibody form: affinity isolated antibody. antibody product type: primary antibodies. clone: polyclonal. purified by: affinity chromatography. species reactivity: human. species reactivity (predicted by homology): nonhuman primates (based on 100% sequence homology). technique(s): western blot: suitable. NCBI accession no.: NP_001008895. UniProt accession no.: Q13619. shipped in: ambient. target post-translational modification: unmodified. Gene Information: human ... CUL4A(8451). Storage Class Code: 12 - Non Combustible Liquids. WGK: WGK 1. Flash Point(F): Not applicable. Flash Point(C): Not applicable.