Journal of Cancer Prevention

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Fig. 4.

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Fig. 4. ERK2-mediated βTrCP1 phosphorylation affects βTrCP1 stability. (A) EGF stimulation induces βTrCP1 phosphorylation. The HEK293T cells transiently expressing HA-βTrCP1 were starved, stimulated with EGF, and harvested as described in Materials and Methods. (B) ERKs Inhibition suppresses βTrCP1 phosphorylation. The HEK293T cells transiently expressing HA-βTrCP1 were starved, stimulated with EGF or the EGF/MEK inhibitor U0126 as indicated, and harvested after 30 minutes. (C) ERK2 knockdown suppresses βTrCP1 phosphorylation. HEK293T cells stably expressing sh-ERK2 cells were transfected with pcDNA3-HA-βTrCP1. (D) Disruption of βTrCP1 and ERK2 interaction prevents βTrCP1 phosphorylation. Cell lysates transiently expressing βTrCP1 in HEK293T cells were utilized. (E) ERK2 reduces the half-life of βTrCP1. The pcDNA3-HA-βTrCP1 was co-transfected with pBIND-Gal4-mock or -ERK2 into HEK293T cells. The cells were treated with cycloheximide (10 µg/mL) and harvested at the indicated time points. The βTrCP1 protein levels were visualized by Western blotting as indicated. (F) ERK2 knockdown attenuates βTrCP1 destabilization. HEK293T cells stably expressing sh-ERK2 cells were harvested at the indicated time point after CHX (10 µg/mL) treatment. The proteins were visualized by Western blotting using specific antibodies as indicated. (G) ERK2 is a upstream kinase of βTrCP1, resulting in reduction of βTrCP1 half-life. (A-D) The phosphorylation of βTrCP1 was visualized by IP/Western blotting as indicated. β-actin was used for equal protein loading as an internal control. IP, immunoprecipitation; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; WCL, whole cell lysates; CHX, cycloheximide; F, F-box domain.
J Cancer Prev 2021;26:174~182 https://doi.org/10.15430/JCP.2021.26.3.174
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