Supplemetary Methods - Recombinant human XIAP (Full length) inhibits DEVD-AFC cleavage in activated cell extracts.

Preparation of Cell Extracts

Materials

• Jurkat E6 cells (ATCC # TIB-152)
• Phosphate Buffered Saline (PBS, pH 7.4)
• Extraction Buffer: 50 mM HEPES-KOH (pH 7.5), 10 mM KCl, 5 mM EGTA, 1 mM MgCl₂, 0.2% CHAPS, 0.2 mM DTT
• Protease inhibitors: Cytochalasin B (Sigma # C6762), Chymostatin (Sigma # C7268), Leupeptin (Sigma # L8511), Antipain (Sigma # A6191), Pepstatin (Sigma # P4265), PMSF (Sigma # P7626)

Methods

1. Pellet cells from the culture media by centrifugation at 1000 x g for 10 minutes at 4 °C.
2. Wash 2 times with PBS and centrifuge as above. Count the cells before the final spin.
3. Add protease inhibitors to the Extraction Buffer immediately prior to use. Final concentrations: 10 µg/mL Cytochalasin B, 2 µg/mL Chymostatin, 2 µg/mL Leupeptin, 2 µg/mL Antipain, 2 µg/mL Pepstatin, 100 µM PMSF, and 1 mM DTT.
4. Solubilize the cells in ice cold Extraction Buffer at a density of 2 x 10⁸ cells/mL.
5. Thoroughly resuspend the pellet by gently pipetting up and down. Incubate on ice for 10 minutes.
6. Pipette 200 µL aliquots into chilled microcentrifuge tubes.
7. Snap freeze in liquid nitrogen and store at ≤ -70 °C. (Note: Freeze immediately at ≤ -70 °C if liquid nitrogen is unavailable to snap freeze.)

Caspase Activation in Cell Extracts

Materials

• Jurkat cell extracts (See Preparation of Cell Extracts)
• Cytochrome c, Bovine heart (Sigma, Catalog # C3131), 2 mg/mL stock in deionized water
• dATP (Sigma, Catalog # D6500), 10 mM stock adjusted to pH 7.5 with KOH
• Recombinant human XIAP (Full length; R&D Systems, Catalog # 822-XF)
• Dilution Buffer: 25 mM HEPES (pH 8.0), 0.1 M NaCl, 1 mM DTT
• Assay Buffer: 10 mM HEPES (pH 7.5), 0.5 mM EGTA, 5 mM DTT, 10% Glycerol
• Substrate: Ac-Asp-Glu-Val-Asp-AFC (DEVD-AFC; MP Biomedicals, Catalog # AFC138), 500 µM stock in DMSO
• EIA/RIA 96-well plate (Costar, Catalog # 3369) or equivalent
• Fluorescence plate reader (Molecular Devices Model # SpectraMax Gemini EM) or equivalent

Methods

Note: All reagents and assay components should be kept on ice until use.

1. Thaw cell extracts and centrifuge at 14,000 x g for 5 minutes at 4 °C. Transfer supernatants to chilled tubes and use within 1 hour.
2. Dilute rhXIAP (Full length; MW: 58 kDa) to various concentrations in Dilution Buffer. Make an initial dilution series of: 2,500, 1,000, 500, 250, 100, 50, 25 and 5 nM. The final concentration range will be 1,000 to 2 nM in a 25 µL total reaction volume.
3. Add 10 µL of cell extract to a tube containing 2.5 µL Cytochrome c, 2.5 µL dATP and 10 µL XIAP.
4. Total (no rhXIAP) and inactive (no rhXIAP, Cytochrome c, or dATP) controls should be run for each assay making up the volume difference with the appropriate buffer.
5. Incubate samples in a 37 °C water bath for 60 minutes.
6. To each well of a 96-well plate, add in the following order, 80 µL Assay Buffer and 10 µL of extracts activated in the presence or absence of added rhXIAP.
7. Start the reaction by adding 10 µL of 500 µM DEVD-AFC (50 µM final concentration).
8. Read at excitation and emission wavelengths 400 and 505 nm, respectively, in kinetic mode for 5 minutes.
9. Derive the 50% inhibiting concentration (IC₅₀) of rhXIAP by plotting RFU/min vs. concentration with 4-PL fitting.

The IC₅₀ for inhibition of DEVD-AFC cleavage in activated cell extracts is typically 40-100 nM.