Fig. 4. Furin colocalizes with proMT1-MMP in intracellular compartments as well as at the plasma membrane of glomerular cells. (A-D) Double labeling of furin (10 nm gold particles) and proMT1-MMP (5 nm gold particles). (A) Both labels are present over the Golgi apparatus (G). Several close colocalizations of the large and small gold particles are visible (arrows). (B) Furin and proMT1-MMP labelings are found in close proximity (encircled) over podocytic vacuoles (V) and the basal surface of podocytes. (C) Higher magnification depicting colocalization of furin and proMT1-MMP (encircled) over the slit diaphragm area and the endothelial fenestrations (D). Bars, 200 nm. (E,F) Immunoprecipitation of total membrane fractions of glomeruli (Memb), treated with DSP (lane 1) or untreated (lane 2), with the anti-proMT1-MMP. The material recovered was separated by 10% SDS-PAGE under reducing conditions and transferred onto nitrocellulose. (E) Immunoblotting with the rabbit anti-furin-N-terminus antibody revealed one band of 98 kDa (arrowhead; lane 1). (F) The same blot shown in E, stripped and reprobed for proMT1-MMP. One band at 63 kDa (arrowhead) is detected independently of chemical crosslinking (lanes 1,2). Notice that, because the immunoprecipitation and the western blotting were conducted with rabbit polyclonal antibodies, there is an immunoglobulin heavy chain band at 55 kDa in each lane. Lane 3, control. Untreated isolated membrane immunoprecipitated with normal rabbit Ig (ctl Ig).

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