Fig. 2. CHMP1 is peripherally associated with the membranes of early and late endosomes. (A) HEK293 cytoplasmic extract was centrifuged at 100,000 g to produce soluble (S100) (lane 1) and membrane-associated (P100) (lane 2) fractions. The membrane pellet was sequentially extracted with 750 mM NaCl (lane 3), 0.1 M Na2CO3 (lane 4), and then the residual was completely solubilized (lane 5). Comparable cell-equivalents from each treatment were resolved by SDS-PAGE and analyzed by western blot for CHMP1. (B) Human 293 cytoplasmic extract was layered on, and centrifuged through, a Percoll gradient. The indicated numbered fractions were collected from the top and analyzed by western blot for CHMP1 and the other early (EEA1) and late (Rab7) endosome, and lysosome (LAMP-1) markers. Note that CHMP1 overlaps well with EEA1, but is also detectable in more dense fractions.
