Supplementary Figure 4: Validation of tetracycline-inducible p53 knockdown and MDM2 knockout cells.
From: Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity
U2OS cells were infected with pLKO tet-on shp53. (a) Western blot showing that doxycycline treatment causes p53 knock-down in 2 days and this effect can be washed off in 5 days. (b) MDM2 was then disrupted by CRISPR. Immunoblotting showing CRISPR disruption (target p53 binding domain) resulted in MDM2 knock-out. (c) Genomic PCR followed by sequencing showing that CRISPR disruption (target p53 binding domain) caused MDM2 knock-out. (d) MDM2 knock-out cells were treated with indicated drugs for 4 hours and phosphorylated p53 (serine 15 and serine 20) are analyzed by western blot.
