p53 protein complex turnover in mitotic cycling cells.

<p>(A) The mCherry tag increases p53-Ch protein in p53 mutant but not wild type mitotic cycling cells. Western blot with anti-p53 to detect endogenous and mCherry-tagged p53 (p53-Ch) protein in 3^rd instar B–D extracts. Lane 1: Wild type. Lane 2: Two copies of untagged, wild type p53 BAC transgene in a p53 null mutant background. Lane 3: One copy p53-Ch in a p53 wild type background. Lane 4: One copy p53-Ch in a p53 null background. Lane 5: Two copies p53-Ch in a p53 null background. * indicates a faint non-specific band at ∼72 kDa. ** indicates a degradation product from p53-Ch. Loading control: Anti-alpha Tubulin. (B) RT-qPCR quantification of p53 mRNA expression levels in <i>p53⁺</i> or <i>p53-Ch; p53^5A-1-4</i> or <i>p53-Ch; p53⁺</i> fly strains. The number of copies for p53 gene is indicated above each genotype. After normalizing to Act5C, the mRNA levels in <i>p53⁺</i> were defined as 1. Error bars represent S.E.M from three independent biological replicates. (C) p53-Ch associates with endogenous p53. RFP-nanobody IP from 3^rd instar B–D extract of the indicated genotypes followed by Western blot with anti-p53. Lane 1,4,7: whole cell extract (WCL), Lane 2,5,8: supernatant (sup) after IP depletion. Lane 3,6,9: IP pellet. * indicates a faint non-specific band at ∼72 kDa. ** indicates a degradation product from p53-Ch. (D) p53 protein level in mitotic cycling B–D cells increases several fold after IR. Western blot with anti-p53 to detect endogenous p53 in 3^rd instar B–D extracts prepared at different time points after IR. Loading control: Anti-alpha Tubulin. (E) Quantification of p53 protein level change in B–D cells at different time points following IR. After normalizing to loading control alpha-tubulin, p53 protein levels from the No IR control was defined as 1. Error bars represent S.E.M from three independent biological replicates. (F) RT-qPCR quantification of p53 mRNA expression level changes following IR. After normalizing to Act5C, p53 mRNA level from No IR sample was defined as 1. Error bars represent S.E.M from three independent biological replicates.