(A and B) Electron micrographs of HeLa cells after either 1 or 5 d of TRP-ML1–specific or control siRNA oligonucleotide transfection

Bars: (A) 2 μm; (B, left) 200 nm; (B, right) 500 nm. (C) Quantitation of the effect of TRP-ML1 knockdown on the formation of storage inclusions. The number of inclusions was calculated using the automated particle counting function of ImageJ. Data are expressed as the number of inclusions per cell slice ± SEM (D) Western blot analysis of siRNA-treated cells expressing siRNA-resistant HA-ML1. HeLa cells were transfected with either control or TRP-ML1–specific siRNA, as indicated. 24 h after siRNA treatment, cells were cotransfected with plasmids encoding GFP and either HA-ML1 (non-siRNA resistant) or HA-ML1 (siRNA resistant). The next day, cells were sorted by FACS analysis to identify GFP-positive (transfected) cells and were subsequently retransfected with the appropriate siRNA and returned to culture for 48 h. Cells were harvested, and 30 μg of total protein was loaded for SDS-PAGE. Samples were transferred to nitrocellulose and immunoblotted to detect endogenous levels of TRP-ML1 (top), HA-ML1 or HA-ML1 (middle), or β-actin (bottom) as a loading control. (E) Quantitation of the effect of HA-ML1 expression on formation of storage inclusions in TRP-ML1 siRNA–treated cells. The number of inclusions was calculated as described in C. The migration of molecular mass markers (in kD) is noted on the left of the blots.<p><b>Copyright information:</b></p><p>Taken from "Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1477-1490.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413042.</p><p>