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. Author manuscript; available in PMC: 2013 Jun 21.
Published in final edited form as: Cell. 2010 Aug 20;142(4):637–646. doi: 10.1016/j.cell.2010.07.014

Figure 4. Caspase-3 and -7 Regulation by the Proteasome.

Figure 4

(A) The Casp3-TevS-2 line was treated with DMSO or 10nM Rap ±5uM MG-132 for 6 hr and cell lysates were blotted with anti-FLAG M2 (left panel, top) or anti-caspase-3 (left panel, bottom) antibodies. The expected processing intermediates detected by each antibody are shown to the right of the immunoblots. The Casp7-TevS-2 line was treated with 10nM Rap for 4 or 8 hr ±500nM PS-341 and immunoblotted with anti-FLAG M2 (right panel) and anti-GAPDH. (B) The Casp3-TevS-2 line was transfected with pcDNA3.1-V5-Ubiquitin for 24 hr and treated with DMSO, 10nM Rap or 10nM Rap + 500nM PS-341 for 4 hr. Cell lysates were immunoprecipitated with anti-FLAG M2 agarose and immunoblotted for covalent ubiquitin with an anti-V5 antibody or with anti-flag to assess caspase-3 recovery. (C) The Casp3-TevS-2 and Casp7-TevS-2 lines were treated with 10nM Rap (blue bars) or 10nM Rap and 5uM MG-132 (orange bars). Caspase activity was measured every 1.5 hr in triplicate (Error Bars±SD). (D) Casp3-TevS-2 and Casp7-TevS-7 cell lines were treated with 10nM Rap ±5uM MG-132 and assayed for caspase activity as above (top) or by staining with GFP-Annexin-V and PI and imaging with fluorescent microscopy (bottom) for indicated time courses in triplicate (Error Bars±SD). See also Figure S4.