(A) HeLa cells were transfected with either nonsilencing control or TRP-ML1–specific siRNA oligonucleotides

Cells were harvested for Western blot analysis after 24 h. Equal amounts of total protein were loaded for SDS-PAGE, as determined by protein assay. Samples were transferred to nitrocellulose and immunoblotted to detect endogenous levels of TRP-ML1 using an antibody directed against the first extracellular loop of the protein. The arrowhead denotes the migration of the cleaved form of TRP-ML1. (B) HeLa cells were transiently transfected with cDNA encoding HA epitope–tagged TRP-ML1 24 h after initial transfection with either control or TRP-ML1–specific siRNA duplexes. After an additional 24-h incubation, cells were solubilized and equal amounts of total protein were immunoprecipitated using anti-HA antibodies. After SDS-PAGE, proteins were transferred to nitrocellulose and probed using horseradish peroxidase–conjugated anti-HA antibody. 10% of the cell lysate was saved before immunoprecipitation and immunoblotted using antitubulin antibody as an additional loading control (bottom). (C) HeLa cells were transfected with either nonsilencing control or TRP-ML1–specific siRNA oligonucleotides and were harvested for Western blot analysis 1, 3, or 5 d after transfection (top), or were retransfected with siRNA duplexes after 2 d and were harvested for Western analysis at 3 or 5 d (bottom). Samples were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted to detect endogenous TRP-ML1. 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>