Fig. 5.
ProBMP4 is trafficked through the TGN and cleaved in an intracellular compartment. (A) Pulse chase analysis of HEK293 cells transfected with DNA encoding BMP4HAmyc, BMP4S2KHAmyc or no DNA (−) suggests that BMP4 is cleaved coincident with or after secretion. Precursor protein and cleavage products were immunoprecipitated from cell lysates and medium at various times postchase as indicated above each lane, deglycosylated by incubation in PNGase and analyzed by SDS-PAGE. Arrows designate nonspecific background bands that are detected in untransfected cells. (B) Endo H-resistant, PNGase-sensitive forms of BMP4 precursor and cleavage products (arrowheads) were detected in the medium but not in cell lysates. Samples were not treated (none) or were treated with Endo H or PNGase as indicated above each lane. Arrows designate nonspecific background bands that are detected in untransfected cells. (C) Blocking TGN export does not lead to accumulation of cleavage products in cell lysates, suggesting that BMP4 is cleaved in a post-TGN compartment. Cells were incubated at either 37°C or 19°C throughout the chase period. Samples were treated with PNGase. Arrows designate nonspecific background bands that are detected in untransfected cells. (D,E) Cell-permeable (RVKR-cmk), but not cell-impermeable (D6R) PC inhibitors, block processing of BMP4, whereas the opposite is observed for Xnr2. Cell lysates and medium from transiently transfected HEK293 cells were collected after treatment with vehicle (DMSO), RVKR-cmk or D6R. Western blots were probed with antibodies specific for the HA tag in the prodomain of BMP4 or Xnr2, or with antibodies specific for the mature domain of BMP4. The two prodomain bands present in the medium of Xnr2-transfected cells correspond to two alternative cleavage sites. Arrow designates a nonspecific background band that is detected in untransfected cells.