Fig 1: Effects of Myc and Skp2 in Ewing sarcoma cell lines using a mouse model.(A) Tumor growth was compared across groups after tumor tissues were transplanted subcutaneously into mice. (B) The expression of p27 and CCNE in mouse tumor tissues was analyzed by immunohistochemistry. Original magnification, × 400; Scale bars: 50 μm. (C) The proportion of immunohistochemically positive cells was quantified. Data represent mean ± SEM of three independent experiments. **, p < 0.01 versus the related control groups. (D) A schematic summary of the study was created, demonstrating that Myc enhances the expression of AP4 and Skp2, ultimately leading to p27 ubiquitination.
Fig 2: AP4 is a target of Myc and decreases p21 expression.(A) The effect of Myc-siRNA on AP4 mRNA expression was evaluated. (B) A time-course analysis showed a correlation between Myc and AP4 protein expression. (C) Downregulation of AP4 led to upregulation of p21. (D) AP4 regulates p21 mRNA expression. The results are represented as the mean ± SEM (n.s., no significance). **, p < 0.01 versus the related control groups. (E) A time-course analysis of AP4 KD showed that p21 expression increased inversely with AP4 levels, while Myc remained unaffected. (F) Changes in p21 expression influenced the phosphorylation of CDK2 and Rb. It was found that p21 reduces phosphorylation of both CDK2 and Rb. (G) The presence of p21 inhibits binding between CDK2 and CCNE.
Fig 3: The influence of p21 on the phosphorylation of CDK2 and Rb protein was examined.Changes in the (A) phosphorylation levels of p27 and Rb protein, and (B) protein levels of Myc, CCNE, CCNA, and CDK2 by the CCNE/CDK2 complex were observed. (C) The effects of the CCNE/CDK2 complex on Rb and E2F1 binding were investigated using immunoprecipitation. The CCNE/CDK2 complex inhibited the binding of Rb to E2F1.
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