Fig. 1. Subcellular localization and interactome of Nephrin and Neph1 in renal podocytes.

A Epitope-localization of the anti-Nephrin/Neph1 ABs used for meAP-MS analysis (further details in Supplementary Fig. 2); inset: Micrograph taken by scanning-EM of glomerular podocytes from rat. Scale bar is 5 µm. Result is representative of at least 100 experiments. B Electron micrographs illustrating the distribution of Nephrin molecules labeled by anti-Nephrin AB a2 (and visualized by gold-particle coupled anti-IgG ABs, black dots) on the extracellular surface (E-face) of freeze-fracture replicas prepared from isolated glomeruli. Image on the right is the frame on the left at expanded scale; FP is foot process, SD is slit-diaphragm. Note that immuno-gold particles for Nephrin are only observed in the slit-area of the SD (arrows). Scale bars are 500 nm. Micrographs are representative of at least four experiments. C Two-dimensional gel separation of CL-47 solubilized membrane fractions prepared from isolated rat glomeruli, Western-probed with ABs against the indicated proteins. Apparent molecular mass (native PAGE, 1st dimension) and molecular weight (denaturing SDS-PAGE, 2nd dimension) are indicated; gel separations were repeated twice with similar results. Note incorporation of all three SD core constituents into high-molecular-mass complexes of ~2 MDa. D Table summarizing the results of the finally annotated constituents of the Nephrin/Neph1 interactome in all six APs with the indicated ABs (Supplementary Fig. 2; AB a3 refers to anti-Neph1 a1). Each column illustrates the results of the indicated target AP controlled by three target-unrelated APs. Color-coding symbolizing evaluation by target-normalized ratios (tnR) as follows: yellow/green: tnR against all thee controls >0.2, white: at least one tnR below the threshold of 0.2, crossed: protein was not identified in the respective target AP. ID refers to the Swiss-Prot entry of the respective protein. MW of all proteins is indicated on the right.