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. 2008 Nov 3;183(3):513–526. doi: 10.1083/jcb.200804048

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Figure 9. — Rab7 depletion impairs processing of cathepsin D. HeLa cells were treated twice at 24-h intervals with inactive siRNA (control; lane 1) or siRNAs to the proteins indicated on top. At 24 h after the second round of siRNA treatment, cells were rinsed with PBS and incubated in serum-free culture medium for 24 h. The medium was collected and precipitated with trichloroacetic acid, and the resulting pellets were dissolved in Laemmli sample buffer. Cell extracts and media samples were analyzed by 4–20% acrylamide gradient SDS-PAGE and immunoblotting with rabbit polyclonal antibody to cathepsin D. Equal amounts of total protein were loaded on each lane. Blots were also probed with antibody to actin as a loading control. The positions of molecular mass markers (in kilodaltons) and of the precursor (pCatD), intermediate (iCatD), and mature (mCatD) forms of cathepsin D are indicated.