IRP activity and TfR1 expression in L-clone and HeLa cells transfected with siRNAs. HeLa cells were transfected with 1.6 μg H-siRNA (H), L-siRNA (L), or the 2 together (H+L) and analyzed 40 hours later, while L-clone was grown in the presence (dox+) or absence (dox-) of doxycycline for 7 days before analysis. (A) Samples of 2 μg of total soluble protein extracts were incubated with a ³²P-labeled IRE H-ferritin probe in the absence or presence of 2% 2-mercaptoethanol (2-β–SH), and the RNA-protein complexes were separated on nondenaturing gel electrophoresis and exposed to autoradiography. (B) Thirty micrograms of cellular extracts were loaded on 12% SDS-PAGE, blotted with a mouse antihuman transferrin receptor antibody (α-TfR1), and developed by ECL. Representative of 3 independent experiments with similar results.