Figure 2. Protease inhibitors increase [⁵⁷Co]Cbl retention in neuronal lysosomes.

Approximately 16×10⁶ SH-SY5Y neurons were metabolically labelled with [⁵⁷Co]Cbl for 48 h. The radiolabelled cells were disrupted using a ball-bearing homogenizer and the lysosomal, mitochondrial and cytosolic fractions were separated using an OptiPrep gradient. Intracellular marker proteins LAMP2 (lysosomal), VDAC1 (mitochondrial), β-actin and MS (cytosolic) were then probed by Western blotting in all fractions. Cells were assessed under standard ‘Control’ culture conditions (A) and after the treatment with 25 μM chloroquine for 48 h (B), 40 μM leupeptin for 48 h (C). The proportional distribution of [⁵⁷Co]Cbl was assessed using a gamma counter and expressed as a percentage of radioactivity in each fraction (D). Positions of molecular mass markers (kDa) are shown on the blots. Fractions are labelled 1–10, from lightest (top of gradient) to heaviest (bottom of gradient). Cyto, cytosol. (A–C) Data are representatives of three independent experiments. (D) Data are means with S.E. represented by the error bars (n=3 experiments for each condition). **P<0.01, ***P<0.0001.