Figure 2.

Impact of CIA knockdown on IRP1, IRP2, and GPAT protein level and cytosolic aconitase activity. A–C, Western blots for endogenous IRP1, IRP2, NuBP2, GPAT, or tubulin were performed on cell lysates as described under “Experimental procedures.” A, wild-type HEK 293 cells were transfected with NT, NuBP2, or Fam96a targeting siRNA and then with or without FBXL5 siRNA as described in the legend to Fig. 1. B, the siRNA transfections were extended such that the wild-type HEK 293 cells were retransfected on day 9 with harvest on day 12 (lanes 1 and 2) or retransfected on days 9 and 12 with harvest on day 15 (lanes 3 and 4). Representative blots from n = 3 experiments are shown. C, wild-type HEK 293 cells were treated as described for A. D, cells were transfected with NuBP2 or Fam96a siRNA, and cytosolic and mitochondrial fractions were obtained after digitonin permeabilization as described under “Experimental procedures” (11). The mean aconitase activities for the cytosolic and mitochondrial fractions were 5.38 and 1.31 milliunits/mg, respectively. Results were determined using a Student's two-tailed t test and are expressed as mean ± S.E. (error bars) for n = 3–6 separate experiments.