Figure 4 | Scientific Reports

Figure 4

From: Asxl1 deficiency in embryonic fibroblasts leads to cellular senescence via impairment of the AKT-E2F pathway and Ezh2 inactivation

Figure 4

Effect of Asxl1 disruption on p27Kip1 phosphorylation and Rb activation. (a) Hypophosphorylation of p27Kip1 by Asxl1 disruption. Phosphorylation of p27Kip1 (Thr157) was monitored by WB using an anti-p-p27Kip1 antibody. (b) Ternary complex of ASXL1, AKT1, and p27Kip1. Lysates were prepared from HEK293 cells with or without IGF-1 treatment and subjected to IP using an anti-p27 antibody. The precipitated proteins were analyzed by WB using anti-ASXL1 and anti-AKT antibody. (c) Effect of Asxl1 disruption on the subcellular localization of p27Kip1. Fluorescence microscopy was performed with MEFs using primary anti-p27 antibody and Alexa 488-conjugated secondary antibody. Hoechst staining was used to visualize chromosomal DNA. (d) Hypo-phosphorylation of Rb. The Rb phosphorylation (Ser807/811) of MEFs (passage 5) from Asxl1 +/+ or Asxl1 −/− mice was monitored daily by WB using an anti-p-Rb antibody. (e,f) Increased Rb binding to the Ccna2 promoter upon Asxl1 deletion. ChIP assays with anti-E2F1 (e) or anti-Rb (f) antibody and primer set for mouse Ccna2 promoter (Supplementary Table 6). Asxl1 +/+ and Asxl1 −/− mice-derived MEFs were treated with IGF-1. Chromatin binding was represented as a percentage of input. Data are the mean ± SD of three independent experiments (*p < 0.05).

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