SBDS loss is not associated with a discrete block in rRNA processing. (A) siRNA against either GAPDH or SBDS was introduced into human skin fibroblasts (GM00038). Cells were lysed 72 hours later and immunoblotted for SBDS and tubulin. GAPDH knock down was confirmed to be greater than 50% by immunoblot densitometry and quantitative PCR (data not shown). (B) GM00038 fibroblasts containing siRNA against GAPDH or SBDS from (A) were metabolically labeled with ³²P-orthophosphate for 75 minutes and chased with 25 mM phosphate for the indicated times in hours. RNA was extracted at the indicated time points, resolved on a 1% agarose/formaldehyde gel, and stained with ethidium bromide to confirm equal loading. (C) RNA from the gel in panel B was transferred to a nylon membrane and analyzed by autoradiography. The positions of the 45S/47S and 32S precursor rRNAs and of the mature 28S, 18S, and 5.8S rRNAs are indicated. (D) GM00038 fibroblasts containing siRNA against GAPDH or SBDS were lysed and fractionated on 10% to 45% sucrose gradients by ultracentrifugation. Absorbance at 254 nM was measured across the gradient, and the positions corresponding to the 40S, 60S, and 80S ribosomal particles are indicated. Results shown are representative of 3 independent experiments.