Gtx108585

Protein separation and western blotting protocols were as described previously^{47 (link)}. Briefly, cells were lysed with RIPA solution, succeeded to SDS-PAGE gel electrophoresis, and the proteins transferred to PVDF membranes. The proteins were probed with anti-CISD2 (1:1,000 dilution; HPA015914, Sigma-Aldrich), anti-V5 (1:2,000 dilution; R960, Invitrogen), anti-GAPDH (1:10,000 dilution; MAB374, Millipore, Billerica, MA, USA), anti-E-cadherin (1:1,000 dilution; #3195, Cell Signaling Technology), anti-vimentin (1:1,000 dilution; #5741,Cell Signaling Technology), anti-ZO-1 (1:1,000 dilution; #5406, Cell Signaling Technology), anti-P21 (1:1,000 dilution; #2947,Cell Signaling Technology), anti-cytochrome C (1:1,000 dilution; GTX108585, GeneTex), anti-caspase 3 (1:1,000 dilution; GTX110543, GeneTex), anti-PARP1 (1:1,000 dilution; GTX100573, GeneTex), anti-EGR1 (1:1,000 dilution; #4153, Cell Signaling Technology), anti-PTEN (1:1,000 dilution; #9188, Cell Signaling Technology), anti-phospho-AKT Ser473 (1:1,000 dilution; (#4060, Cell Signaling Technology), and anti-AKT1 (1:1,000 dilution; #2938, Cell Signaling Technology) antibodies, and then further probed by horseradish peroxidase-conjugated secondary antibodies.

The antibodies used in this study are as follows: anti-active-caspase-3 rabbit polyclonal antibody (G7481, Promega), anti-Bax mouse monoclonal antibody (6A7, Sigma-Aldrich), anti-hBid rabbit polyclonal antibody (AF846, R&D, Minneapolis, MN), anti-Bak mouse monoclonal antibody (MAB8161, R&D), anti-HA tag antibody (HA.11/16B12, Covance Inc., Princeton, NJ), anti-Green fluorescent protein monoclonal antibody (mFX73, Wako, Osaka, JAPAN), anti-HIF1 alpha Rabbit polyclonal antibody (GTX127309, GeneTex, Irvine, CA) and anti-cytochrome-c Rabbit polyclonal antibody (GTX108585, GeneTex).