Mislocalization and defective glycosylation of mutant Galnt3.

<p>(<b>A</b>) COS-7 cells were transiently transfected with either EGFP-wild-type, WT (Trp589), or EGFP-mutant (Arg589) constructs, and counterstained with anti-GM130 antibody, which immunostains the Golgi apparatus (red), or anti-PDI antibody, which immunostains the ER (red). DAPI was used to stain the nucleus (blue). WT Galnt3 co-localizes with GM130, but not PDI (data not shown), thereby revealing that it is targeted to the Golgi apparatus. However, the mutant Galnt3 co-localizes with PDI and is predominantly found in the ER. (<b>B</b>) Western blot analysis of kidney homogenates using anti-GALNT3 antibody, revealed that protein lysates from WT littermates, and <i>Tcal</i>/+ mice had three immunoreactive products (a, b and c) whereas those from <i>Tcal/Tcal</i> mice had only two products (b and c). PNGase F treatment resulted in loss of the largest Galnt3 product (band a) observed in the lysates from WT littermates, and <i>Tcal</i>/+ mice, indicating that these were glycosylated products.