Figure 3

Confirmation of RNA-protein binding on microarrays with reciprocal pull-down assays for Staufen1 with TP53 mRNA. (A) Stau1-HA protein pulls down TP53 mRNA in vitro after immunoprecipitation with HA Mab; immunoblots to HA-tagged Stau1 verifying Stau1 precipitation are shown on the left panel. (B) Stau1-HA protein pulls down TP53 mRNA in vivo, but not HRAS and GAPDH control RNAs after immunoprecipitation with HA Mab; immunoblots to HA-tagged Stau1 verifying Stau1 precipitation from cell extracts are shown on the left panel. (C) Pull-down of biotin labeled human TP53 mRNA in vitro, but not HRAS or Lac Z mRNA precipitates associated Stau1-HA protein; densitometry quantification of the immunoblots shown on the right panel. (D) Schematic diagram of TP53 mRNA constructs. Numbering corresponds to the first nucleotide following the termination codon, defined as 1. Signs (“+” and “–“) represent ability or failure of Stau1-HA to bind TP53 RNA constructs. (E) Pull-down of biotin labeled human TP53 mRNA deletion constructs in vitro, followed by western blot analysis of the associated Stau1-HA protein; densitometry quantification of the immunoblots shown on the right panel. (F) TP53 RNA decay in Primary Fibroblasts after actinomycin D treatment. (G) Comparison of full-length TP53 and TP53-ORF (lacking Staufen 1 interaction domain) RNA decay in TP53 negative H1299 cells after actinomycin D treatment.