Figure 3. FPC is cleaved in vivo.

(a) Schematic representation of full length FPC (left) and cleaved FPC (right) with proposed cleavage sites indicated by red arrows. Cleavage of full length FPC at a probable proprotein convertase site produces a large extracellular domain (PECD) that is tethered to a C-terminal fragment (PTM) by disulfide bridges. The PTM is further cleaved by a γ-secretase to release an intracellular C-terminal fragment (ICD) containing the putative nuclear localization signal (NLS). IPT: Ig like, plexins, transcripton factor domain PbH1: Parallel beta-helix repeat, TM: transmembrane segment (b) Western blots were prepared from total lysates derived from renal tubules isolated at P7. Blots were probed with anti-HA (left panel) or mFPC-ct (right panel). Anti-HA detects full-length FPC (FPC-HA, ~500KDa) and another FPC related band at ~55KDa that corresponds with the predicted PTM (PTM-HA). These bands were present only in Pkhd1^Flox67HA but not in Pkhd1^wt or Pkhd1^Δ67 tubules. The mFPC-ct antibody also detected full-length FPC and the PTM in Pkhd1^Flox67HA and Pkhd1^wt tubules but not in Pkhd1^Δ67 tubules. (c) Western blots of total lysates prepared from Pkhd1^Flox67HA and Pkhd1^Δ67 adult kidney (K) and pancreas (P), probed with anti-HA. Anti-HA detects FPC-HA and PTM-HA only in Pkhd1^Flox67HA tissues. The PTM is absent in pancreas. (d) Western blots prepared from total adult kidney lysates run on 4–12% Bis-Tris acetate gels to enhance detection of small proteins. Anti-HA detects FPC-HA and a small 17KDa fragment harboring that corresponds with the size of the cleaved C-terminal ICD product (ICD-HA). The PTM isn’t visualized because it is presumably obscured by a non-specific band just above 50KDa