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. Author manuscript; available in PMC: 2009 Sep 9.
Published in final edited form as: Biochemistry. 2008 Aug 15;47(36):9678–9687. doi: 10.1021/bi800699v

Figure 2. α-Syn degrading activities in subcellular fractions are proportional to their cathepsin D content.

Figure 2

A. Subcellular fractions (56 μg of protein) from 3D5 cells isolated after 7 days in TetOFF medium were incubated in cathepsin D buffer pH 4 at 37°C. B. Lysosomal fractions from the same preparation described in A were analyzed at shorter incubation times. C. Subcellular fractions (5 μg of protein) from 3D5 cells cultured in TetON medium (background α-syn expression in these cells was not detectable during the time of exposure shown in the Figure) were incubated with 2 μg of recombinant human wild-type α-syn in cathepsin D buffer. Overnight (O/N) samples shown in Panels A and C, and 2h samples in B were incubated without inhibitors (−), with pepstatin A (P), leupeptin (L), or a cocktail of protease inhibitors (PI). Aliquots at the time points indicated in the Figure were analyzed by Western blot using syn-1 antibody. The blot shown in panel A was also probed with a cathepsin D antibody. The positions of 12 kDa and 10 kDa α-synΔC species are indicated with broken and full arrows, respectively. The empty arrow in panel C indicates the position of 11 kDa α-synΔC species. Representative blots from four experiments are shown.