Differential centrifugation of PBMC lysate yields fractions that are highly enriched in endolysosomes. (A) Enrichment of endolysosomes in fractions recovered after differential centrifugation was assessed by western blot. PBMC lysate from which endolysosomes were extracted (lane 1), fraction enriched in endolysosomal proteins LAMP1 and Cathepsin S (lane 2). (B) Isolated endolysosomal (light grey) and cytosol depleted of lysosomal (dark grey) fractions were assayed for activity of cathepsins, aminopeptidases and chymotryptic activity of proteasome in the presence of inhibitors Epoxomicin (Epox) for proteasome, Bestatin (Best) for aminopeptidase, E64 for cathepsins and EDTA as a metal chelator. Mean and SD of replicates from two different donors are plotted. (C) Acid phosphatase assay was performed to confirm enrichment of endolysosomes in different fractions. Figure representative of acid phosphatase activities in one fractionation experiment done in triplicates. Mean and SD are plotted. (D) Two HIV Gag peptides, B57KF11 (KAFSPEVIPMF) and B27KK10 (KRWIILGLNK) were subjected to degradation by equivalent amounts of purified cytosol (blue) or endolysosomes (red). An aliquot of the reaction mixture was stopped at periodic intervals and the percentage of original peptide remaining was determined by HPLC. The half-life of the peptide in both conditions was calculated (36 min in cytosol and 8 min in endolysosomes for B57KF11 and more than 60 min in cytosol and 38 min in endolysosome for B27KK10). Figure 1D is representative of two replicates.
Vaithilingam et al. BMC Cell Biology 2013 14:35 doi:10.1186/1471-2121-14-35