Rabbit polyclonal antibody for caspase 3

Detection of cleaved caspase-3 induced by DHMEQ and melphalan was performed by western blot analysis. Antibodies used in these experiments were as follows: Rabbit polyclonal antibody for cleaved caspase-3 (Asp175) (Cell Signaling Technology, Inc.), rabbit polyclonal antibody for caspase-3 (Cell Signaling Technology, Inc.), and tubulin (Santa Cruz Biotechnology, Inc.). Cells were lysed in lysis buffer (RIPA). Cell lysates were subjected to electrophoresis on an SDS polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were blocked with TBS containing 0.05% Tween and 5% nonfat milk at 4˚C overnight. Subsequently, they were incubated for 1 h with anti-caspase-3 or anti-tubulin antibodies. The membranes were then incubated for 1 h with peroxidase-labeled anti-rabbit or anti-mouse secondary antibody and developed using an enhanced chemiluminescence system. ImageJ software-based analysis was used to quantify the intensity of the bands obtained by the Western blot assay.