Figure 3.

Glomeruli of Rhpn1 ko mice do not express Rhophilin-1, and GFP can be used as a surrogate of its expression. (A) Glomerular-restricted expression of Rhophilin-1 in wild-type mice is absent in kidneys of Rhpn1 ko mice, which correspondingly display GFP expression confined to glomeruli. (B) Immunoblotting of glomerular lysates confirms expression of Rhophilin-1 in wild-type (wt) and heterozygous (het) mice and its deletion in Rhpn1 ko animals. GFP is expressed in a reciprocal manner. Molecular mass markers (kilodaltons) are indicated to the left. (C) Live phase contrast and GFP (green) images of purified glomerular suspensions isolated from Rhpn1 ko mice show the purity of glomerular preparations as well as GFP-restricted expression to podocytes and their processes. Magnetic beads (4.5 μm in diameter) are observed as opaque spheres within glomeruli. (D) Primary cultures of glomeruli isolated from Rhpn1 null mice give rise to WT1-expressing outgrown podocytes that stain positively for GFP (arrows). Outgrown cells that do not express WT1 do not express GFP (arrowheads). DAPI, 4′,6-diamidino-2-phenylindole. Scale bars, 100 μm in A; 50 μm in C, left panels; 10 μm in C, right panels; 20 μm in D.