Association of IRP1C437S with the polysome-bound ferritin mRNA. (A) The experimental procedure; cell lysates obtained from H1299 cells grown at high density in the presence or absence of tetracycline were subjected to either Western blotting with ferritin and β-actin antibodies (B), or qPCR with H-ferritin, TfR1, and GAPDH specific primers (C), or ultracentrifuged for 1 h to isolate the heavy polysomes from the monosomes. The polysomal pellet was subjected to immunoprecipitation with a FLAG antibody. Ninety pecent of the immunoprecipitate was used for RNA extraction and reverse transcription PCR with H-ferritin, TfR1, and GAPDH-specific primers (D), and 10% of the immunoprecipitate was subjected to Western blotting with a FLAG antibody to document the presence of FLAG-tagged IRP1C437S in the immunoprecipitate (E).
