Expression and puriﬁcation of recombinant proteins in bacteria The pET15b expression plasmid encoding full-length human BID was pro- vided by X.-M. Yin (University of Pittsburgh School of Medicine, Pittsburgh, PA), the pET23dwHis expression plasmid containing full-length mouse BID was provided by X. Wang (University of Texas Southwestern Medical Center, Dallas, TX), and G.S. Salvesen (The Burnham Institute of Medical Research, Pittsburgh, PA) provided the pET23b expression plasmid containing full-length human caspase-7. Expression plasmids were transformed into the bacterial E. coli strain BL21 (DE3) and were expressed and afﬁnity puriﬁed as previously described (Hill et al., 2004), with the exception that caspase-7 was induced for 40 min to minimize the production of the processed form of this protease. Full-length CrmA was cloned into the expression plasmid pGEX4TK2 (GE Healthcare) in frame with the GST coding sequence at the N terminus. GST-CrmA was expressed in E. coli DH5α and afﬁnity puriﬁed using glutathione–Sepharose as previously described (Slee et al., 1999). Active site titration of recombinant granzymes 1 μM GST-CrmA was activated by incubation with 500 μM dithiothreitol for 10 min at 37°C. Active CrmA was then incubated with human or mouse GzmB for 10 min at 37°C. Residual GzmB activity was then determined by diluting samples into PBS containing 500 μM Ac-IETD-AFC and measuring hydrolysis of the latter peptide by ﬂuorimetery, as described in Fluorometric assays. Kinetic analysis of substrate proteolysis by granzymes Typically, 3–4 μg of recombinant human or mouse BID was incubated for 2–3 h at 37°C with varying concentrations of human or mouse GzmB in 10 μl reaction volumes. Human recombinant pro–caspase-7 was treated in an identical manner, but with 10 μM zVADfmk added to inhibit caspase autoprocessing. Reaction products were separated by SDS-PAGE and visualized by Coomassie blue staining. Densitometry was performed using ImageJ software (National Institutes of Health; http://rsb.info.nih.gov/ij/) and catalytic rates were calculated using these data. Cell-free reactions Cell-free cytosolic S-15 extracts were generated from Jurkat and J774 cells, as previously described (Martin et al., 1996; Slee et al., 1999). Typically, human and murine extracts were normalized to 10 mg/ml total protein content, and then diluted twofold in WCEB (20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 100 μM PMSF, 10 μg/ml leupeptin, and 2 μg/ml aprotinin). GzmB and zVADfmk were added to the reactions at ﬁnal concentrations of 6–100 nM and 10 μM, respectively. Reactions were incubated for 1–2 h at 37°C to facili- tate GzmB-mediated proteolysis. Cell-free reactions were then assessed by immunoblotting or ﬂuorimetric assay. Fluorimetric assays Reactions containing Jurkat or J774 cell-free extracts (normalized for total protein content) were typically assembled in a ﬁnal volume of 10 μl. After a 30-min incubation at 37°C, 2.5-μl samples were diluted to a ﬁnal volume of 100 μl in WCEB containing 50 μM Ac-DEVD-AFC. To determine the activity of recombinant GzmB, protein was diluted in 30 μl PBS containing 500 μM Ac-IETD-AFC. All samples were measured in an automated ﬂuori- meter (Spectraﬂuor Plus; TECAN) at wavelengths of 430 (excitation) and 535 nm (emission). Clonogenic assays For clonogenic assays on Jurkat and J774 cells, a 3-ml bottom agar layer was poured on 6-cm culture dishes containing RPMI with 20% fetal calf serum, 1% L-Glutamine, 2 mg/ml sodium bicarbonate, 0.001 mg/ml folic acid, 10% cell-conditioned medium, and 0.5% Noble agar, and this was allowed to set for 30 min. A top layer, containing a 100-μl aliquot of cells, was added to 4 ml of the same medium containing 0.33% Noble agar (prewarmed to 37°C), and this was poured over the bottom layer and allowed to set. The agar layer was then overlaid with 2.5 ml of the medium (without agar) and incubated at 37°C for 3–4 wk. Plates were washed brieﬂy in PBS, pH 7.2, and stained for 30 min with a solution of 0.05% crystal violet in PBS, pH 7.2, containing 20% methanol. For clonogenic assays using MCF-7 cells, cells were plated on 6-cm culture dishes with RPMI, 20% fetal calf serum, 1% L-Glutamine, and 10% cell-conditioned medium and incubated at 37°C for 1 wk. Plates were then washed brieﬂy in PBS, pH 7.2, and stained for 30 min with a solution of 0.05% crystal violet in PBS, pH 7.2, containing 3% paraformaldehyde. Gzmb/SLO-killing assays Typically, SLO and GzmB were added to cells (at concentrations indicated in the ﬁgure legends) at 106 cells/ml. For clonogenic assays, 30,000 cells were treated in a ﬁnal volume of 30 μl in 96-well U-bottomed plates, fol- lowed by incubation at 37°C for 45 min. SLO was deactivated by diluting cell cultures 1:10 with RPMI containing 10% FCS, and cells were trans- ferred to 96-well ﬂat-bottomed plates for further observation. Cell death was subsequently assessed by ﬂow cytometry using annexin V/propidium iodide (PI) staining (Martin et al., 1995). Image acquisition and analysis The cell images presented in Fig. 5 A, Fig. 6 A, Fig. 7 A, and Fig. S2 A were taken on an inverted microscope (IX71; Olympus) using a 40× objective, and images were captured using a Colorview II camera (Soft Imaging System) equipped with Analysis image acquisition software. All images were acquired at room temperature. Images of crystal violet– stained colony formation assays presented in Fig. 6 D, Fig. 7 E, and Fig. S2 D were captured on a digital camera (CoolPix 5000; Nikon) and imported into Photoshop (Adobe) for uniform brightness and contrast adjustment. Densitometric analysis of the Coomassie blue–stained gels presented in Fig. 3 (A and B) was performed using ImageJ software. All Western blot images were scanned from x-ray ﬁlm using a scanner (Epson Perfection) and Photoshop software. All ﬁgures were prepared using Page- Maker 7.0 software (Adobe). Online supplemental material Fig. S1 shows the cytochrome c/dATP–induced processing of caspase-8 in mouse J774 cell-free extracts. Fig. S2 shows that caspase-3 deﬁciency is not sufﬁcient to afford clonogenic rescue from human or mouse GzmB. Online supplemental material is available at http://www.jcb.org/cgi/ content/full/jcb.200612025/DC1. We thank Professor Winfried Wels for provision of the P. pastoris strain expressing human GzmB. S.J. 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