CD63 expression is posttranscriptionally regulated by the IRE-IRP system. (A) Schema of the size and locations of the 5′-IRE domains in CD63, FTH, and FTL mRNAs. (B) CD63 IRE-containing mRNA and non-IRE mRNA were biotinylated and mixed with a total protein extract from IMR90SV cells followed by pulldown using streptavidin beads. Input and pulldown proteins were analyzed by using anti-IRP1 and anti-IRP2 antibodies. Upon equal exposure of the input and pulldown, the percentage of IRP pulled down from the input was 2.5%. (C) Quantification of IRP1 and IRP2 intensity in panel B. (D) Purified GST-IRP1 or GST-IRP2 was mixed with biotinylated FTH IRE, CD63 IRE, or non-IRE RNA (see “Methods” for details). Samples were loaded onto a native 6% TBE-polyacrylamide gel, transferred onto nylon membrane, and stained with streptavidin-horseradish peroxidase. (E) Quantification of shifted bands in panel D. Results are mean ± SEM (3 experiments). Significance was determined by Student t test. ***P < .001; ****P < .0001.