Anti klf12

Cells were harvested and lysed using lysis buffer (Cell Signaling Technology) containing protease inhibitors (Sigma) and phenylmethylsulfonyl fluoride (PMSF) (Sigma Chemical Co., St Louis, MO, USA). Equal amounts of protein were separated by 10% SDS-PAGE and then transferred to Immobilon-P Transfer Membranes (Millipore Corporation, Bedford, MA, USA). The membranes were pre-blotted in 5% skim milk prior to incubation in 1% skim milk containing primary anti-Sp1 (1:500; Millipore Darmstadt, Germany), anti-KLF12 and anti-XIAP (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-cleaved-PARP, anti-cleaved-caspase3 (1:1000; Cell Signaling Technology, Inc., Danvers), anti-DDK (1:1000) (OriGene Technologies, Rockville, MD) and anti-β-actin (1:10000; Sigma-Aldrich, St. Louis, MO) antibodies overnight. The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Amersham) and visualized using ECL^TM Western Blotting Detection Reagent (Amersham). Images were captured by Fuji Medical X-Ray Film (Fuji) and developed by the Fuji system. The Human Apoptosis Array Kit (R&D Systems, Inc., USA) was used based on the manufacturer’s instructions.