Fig. 2. BRO1 is required for sorting CPS via the MVB pathway. (A) Fluorescence microscopic localization of GFP-CPS and FM 4-64. Arrows indicate class E compartments. (B) Newly synthesized CPS was immunoprecipitated from lysates of cells that had been pulse-labeled with [³⁵S]methionine/cysteine for 10 minutes then chased in non-radioactive medium for 0 or 30 minutes. Immunoprecipitates were resolved by SDS-PAGE and examined by fluorography. (C) Immunoprecipitates of CPS were resolved by SDS-PAGE, transferred to nitrocellulose, and examined by western blotting using anti-ubiquitin antibodies. Note that CPS is normally differentially modified by the addition of two or three oligosaccharide moieties and is, therefore, observed in C as a doublet (Spormann et al., 1992); however, in B, the immunoprecipitates were treated with endoglycosidase H in order to facilitate the detection of precursor CPS (pCPS) and mature CPS (mCPS).