Journal of Cancer Prevention

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Fig. 3.

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Fig. 3. Determination of ERK2 binding sites of βTrCP1. (A) Amino acid homology of putative ERK2 docking sites (PEDS). Amino acids for two putative ERK2 docking sites were compared to different animal species as indicated. The numbers at the top indicate the position of amino acids. The red color indicates amino acids for putative ERK2 docking sites. (B) Mutation strategies to construct mutant PEDS 1 and 2. The mutants were recombined into pcDNA3-HA plasmids. (C) Confirmation of βTrCP1 PEDS 1 and 2 for ERK2 binding. The pcDNA4-HisMAX-ERK2 and each of the constructs in (B) were co-transfected as indicated. The interaction between ERK2 and each of βTrCP1 mutants was visualized by IP/Western blotting as indicated. (D) The constructs in (B) were transfected into HEK293T cells. Endogenous Cullin 1 binding to βTrCP1-mtPEDS1 and -mtPEDS2 were visualized by IP/Western blotting as indicated. WT, wildtype; D, D domain; F, F-box domain; mtPEDS, mutant putative ERK docking site; Xp, Xpress tag; ERK2, extracellular signal-regulated kinase 2; IP, immunoprecipitation; HA, hemagglutinin; WCL, whole cell lysates.
J Cancer Prev 2021;26:174~182
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