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. 2018 Jun 6;8(13):3517–3529. doi: 10.7150/thno.24401

Figure 5.

Figure 5

p53 silencing partially rescues the effects of HUWE1 deletion. (A) Western blotting for the indicated proteins in teton-shTP53 lentivirus-infected HUWE1-wild type cells and HUWE1-null cells; TP53 knockdown was induced by doxycycline (DOX). β-actin was used as a loading control. (B) Growth curves for the indicated cells. The data are presented as the means ± SD (*, P < 0.05, unpaired t-test). (C) Soft agar colony formation assay of the indicated cells. Quantification of colony numbers is shown. The data are presented as the means ± SD (**, P < 0.01, unpaired t-test). (D) Proteins were extracted from the HUWE1-intact clone A549AA1. Co-immunoprecipitation assays were performed to assess the interaction between HUWE1 and p53 using the p53 antibody. Rabbit IgG was used as a negative control. (E) In vivo ubiquitination assays to evaluate the amount of ubiquitinated p53 in HUWE1 wild type cells and HUWE1-null cells. The cells were treated with 5 μM MG-132 for 6 h, then lysed, and subjected to immunoprecipitation with the p53 antibody. Ubiquitinated p53 was detected by immunoblotting with an anti-ubiquitin antibody.