Fig. 5. Resistance of NPP3 to Lubrol extraction is acquired early, independently of targeting. MDCK cells stably expressing NPP3-WT or NPP3-AAASLLAP were labeled for 1 hour with [³⁵S]Met-[³⁵S]Cys. Cells were lysed with Lubrol WX and separated on sucrose gradients. Each fraction was subjected to immunoprecipitation with anti-NPP3 antibody, then immunoprecipitates corresponding to the DRM and soluble fractions (Sol) were separately pooled, and treated (+) or not treated (–) with endo H, separated by SDS-PAGE electrophoresis and analyzed by fluorography. The mature (1), high-mannose (2) and deglycosylated (3) forms are indicated by arrows. The mature forms of both NPP3-WT and NPP3-AAASLLAP (band 1) migrate slightly faster after endo H treatment because not all glycan chains are complex glycosylated (Maurice et al., 1994).