Fifty micrograms of cell lysates were incubated with 200 nmol/L Ac-DEVD-AFC (for caspase-3), Ac-IETD-AFC (for caspase-8), and Ac-LEHD-AFC (for caspase-9) in a reaction buffer containing 20 mmol/L HEPES, pH 7.4, 100 mmol/L NaCl, 10 mmol/L DTT, 0.1% CHAPS, and 10% sucrose at 37°C for 1 h.
Five micrograms of cell lysates were added to wells containing pre-adsorbed STAT consensus oligonucleotides (5'-TTCCCGGAA-3').
Seven- to eight-hundred microframs of cell lysate were incubated with 2 µg of anti-Myc antibody (Santa Cruz Biotechnology, Santa Cruz, CA) with gentle rotation at 4°C overnight.
Twenty-five microframs of cell lysate were loaded and resolved by SDS-PAGE as previously described.
Fifty micrograms of cell lysate was resolved by SDS-PAGE and transferred to nitrocellulose membranes (0.22 μm, Millipore, Bedford, MA).
Twenty micrograms of cell extracts was loaded into 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blot.
Four micrograms of cell lysate were loaded per lane on a MiniProtean TGX™ pre-cast gel (Biorad) along with the Precision plus protein Kaleidoscope™ (BioRad) molecular mass markers.
Ten micrograms of cell lysate from each sample were used for SDS-PAGE (Bio-Rad Landratories, Richmond, CA), and Western blots were performed according to the protocol we described previously [ 16].
Thirty micrograms of cell lysate from each sample were used for SDS-PAGE (Bio-Rad Landratories, Richmond, CA), and Western blots were performed according to the protocol as described previously [ 11].
Sixty micrograms of cell lysate were separated by SDS/PAGE on a 10% polyacrylamide gel and transferred to nitrocellulose membrane (Schleicher &Schuell).
