Stability of B27KK10 peptide in the presence of different inhibitors. (A) The stability of an HIV peptide, B27KK10 in crude PBMC lysate in various pH buffers was determined using a degradation assay at pH 3.0 (red), pH 4.0 (orange), pH 5.0 (purple), pH 6.0 (green), pH 7.0 (blue), pH 8.0 (black). The amount of remaining peptide was monitored by RP-HPLC during 60 min. (B) As a control, the peptide was incubated in the buffer without crude lysate. (C) When using inhibitors, the crude lysate was pre-incubated with inhibitors for all cysteine cathepsins (E64), Cathepsin S (Cat S), D (Cat D), K (Cat K), B (Cat B), aminopeptidase (Amino) and proteasome (Prote) for 30min at RT before addition of the peptide. An aliquot of the reaction mixture was stopped at periodic intervals and the percentage of original peptide remaining was determined by HPLC. Based on a one-phase exponential decay, the stability rate of the peptide was calculated. The stability rate of the peptide without any inhibitor was assigned a value of one and the fold change with inhibitors was determined. The average stability rate from three different experiments using PBMC lysate from three different donors was plotted.
Vaithilingam et al. BMC Cell Biology 2013 14:35 doi:10.1186/1471-2121-14-35