Journal of Cancer Prevention

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Fig. 1.

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Fig. 1. ERK2 and βTrCP1 are components of the Cullin 1 containing SCFβTrCP1 complex. (A) Mammalian two-hybrid assay screening. Mammalian two-hybrid recombinant plasmids including pACT-VP16-bβTrCP1, pG5-luciferase reporters, and each of pBIND-Gal4-kinases (as indicated) were transfected into HEK293T cells with a molar ratio 1:1:1. Relative luciferase activity was converted by comparison to luciferase activity obtained from pACT-VP16-βTrCP1/pBIND-Gal4-mock/pG5-luc. Equal transfection was normalized by Renilla luciferase activity obtained from each of pBIND-Gal4-kinase expression vectors. (B) Confirmation of βTrCP1 and ERK2 interaction obtained through IP. pBIND-Gla4-MAPKs (as indicated) and pcDNA3-HA-βTrCP1 were co-transfected into HEK293T cells. The interaction of ERK2 and βTrCP1 was visualized by IP/Western blotting as indicated. (C) Confirmation of ERK2 and Cullin 1 interaction. pcDNA3-Myc-Cullins and pcDNA4-HisMAX-ERK2 were co-transfected into HEK293T cells. The interaction of ERK2 and each of the Cullins was visualized by IP/Western blotting as indicated. CDK, cyclin-dependent kinase; DAPK, death-associated protein kinase; GRK5, G protein-coupled receptor kinase 5; LCK, lymphocyte-specific protein-tyrosine kinase; NEK6, NIMA-related kinase 6; CSK, C-terminal SRC kinase; STK16, serine/threonine-protein kinase 16; TOPK, lymphokine-activated killer T-cell-originated protein kinase; PAK2, p21 protein-activated kinase 2; SGK, serum/glucocorticoid-regulated kinase; ERK, extracellular signal-activated kinase; JNK2, c-Jun N-terminal kinase 2; IP, immunoprecipitation; HA, hemagglutinin; WCL, whole cell lysates.
J Cancer Prev 2021;26:174~182
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