FIGURE 8:

Uroplakins are processed through the ER-Golgi-TGN pathway in the Snx31-KO urothelia. (A) Assessing uroplakin trafficking through the ER-Golgi-TGN pathway based on their glycosylation and proteolytic processing status. Crude membrane proteins from the wild-type (lanes 1–3) and Snx31-KO (lanes 4–6) mouse urothelia were incubated with buffer (lanes 1 and 4; control or “C”), Endo H (“H,” lanes 2 and 5; asterisks in panel a mark the added Endo H enzyme), or PNGase F (lanes 3 and 6; “F”). The electroblotted samples were stained with (a) Coomassie Blue (CM), or immunoblotted with antibodies to (b) Snx31, (c) UPIa, (d) UPII, (e) UPIb, or (f) UPIIIa. Note that, like in the wild-type urothelium, the glycans of UPIa of the Snx31-KO are Endo H sensitive, those of UPIb and IIIa are PNGase F sensitive, and that the 32 kDa pro-UPII is cleaved by the TGN-associated furin to yield the 15 kDa mature UPII. Five wild-type and 15 Snx31-KO mice were used for protein collection. (B) Double staining of KO urothelial sections for UPIb (green) and Sec23a, an ER marker. (C, D) Double staining of the Wt and Snx31-KO urothelial sections for uroplakin Ia (green) and ER stress markers calnexin (C2, D2). (E, F) Double staining of KO urothelial sections for UPIb (green) and (E) GM130, a Golgi marker, or (F) TGN38, a TGN marker. Scale bars = 10 µm in B–F.