Figure 5
FPC positive vesicles contain NEDD4 interacting protein NDFIP2. (a) Microtube containing pellet of vesicles isolated from HEK cells expressing PKHD1-Myc using either anti-HA or anti-Myc coupled beads. A small pellet is visible in the anti-Myc sample but not in the anti-HA negative control. (b) Immunblot of vesicles isolated using either anti-Myc (lanes 1,4) or anti-FLAG (lane 2) coupled beads from HEK cells with either vector control (lanes 1,2) or expressing PKHD1-Myc (lane 4) and analyzed with antibodies against Myc, NDFIP2, SMURF1, SMURF2 and NEDD4-2. Lane 3 contains 20 μg of total lysate of the PKHD1-Myc sample. Total protein quantity was accessed by Coomassie brilliant blue staining (CBB). (c) Immunblot of vesicles isolated using either anti-HA (lanes 1, 4) or anti-FLAG (lane 2) coupled beads from fresh kidneys of wild type (lanes 1, 2) or Pkhd1 HA-Flox mice (lane 4) and probed with antibodies against HA, Ndfip2, Smurf1, Smurf2 and Nedd4-2. Lane 3 contains 20 μg of total lysate of the Pkhd1 HA-Flox sample. Total protein quantity was assessed by Coomassie brilliant blue staining (CBB). Anti-HA detects full length Fpc. The full-length blots of cropped images in b and c are included in Supplementary Fig. S9. (d–f) Confocal analysis of kidney (d), liver (e), and pancreas (f) from Pkhd1 HA-Flox (top panels in each) or wild type mice (bottom panels in each) for Fpc (HA) (green), Ndfip2 (red), DAPI (blue) and markers for collecting duct (aquaporin 2, Aqp2) or epithelial cells (cytokeratin 19, Ck19). Merged images are on far right. Scale bars, 20 μm in “d” and “e” and 10 μm in “f”. (g) Kidney specimens from human control (control) and two different ARPKD donors (ARPKD1, ARPKD2) stained for DBA (grey), LAMP1 (green), NDFIP2 (red) and DAPI (blue). Scale bars, 10 μm.
