Semi dry fast blot apparatus

Wholecell lysates were obtained from HL-60R cells by lysis with RIPA buffer (Santa Cruz Biotechnology Inc., Dallas, TX, USA). A mass of 25 µg of proteins were separated using a 10% SDS-PAGE acrylamide gel. Nitrocellulose membrane transfer (Amersham, Pharmacia Biotech, Milan, Italy) was done using a semi-dry fast blot apparatus (Bio-Rad, Milan, Italy). After saturating the membranes with BSA 5% (w/v) in PBS-0.1% (v/v) Tween 20 for 1 h, the filters were incubated with primary antibodies against GAPDH (1: 20,000; Sigma-Aldrich Srl, Milan, Italy), survivin (1: 2000, Abcam Limited, Cambridge, UK), Bcl-2 (1:1000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), XIAP (1:500; Cell Signaling Technology, Inc. Danvers, MA, USA), and P-gp (1:100, Invitrogen, Milan, Italy). Using an advanced chemiluminescence detection kit, the SuperSignal West Femto Substrate of maximum sensitivity (Thermo Scientific Life Technologies Italia, Monza, Italy) and the Versa DOC imaging system (Laboratori BioRad, Milan, Italy) the hybridization was analyzed. Immunoblots were quantified by densitometry, normalized against GAPDH values and expressed as relative protein levels.

The whole-cell lysates were obtained from HL-60R cells using radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and 25 µg protein was subjected to 10% SDS-PAGE and transferred to nitrocellulose membrane (Amersham, Pharmacia Biotech, Milan, Italy) using a semi-dry fast blot apparatus (Bio-Rad, Milan, Italy). Membranes were blocked with 5% (w/v) BSA in PBS-0.1% (v/v) Tween 20 for 1 h and then filters were incubated with primary antibodies raised against GAPDH (1:20,000; Sigma-Aldrich Srl, Milan, Italy.), XIAP (1:500; Cell Signaling Technology, Inc. Danvers, MA, USA), survivin (1:2000, Abcam Limited, Cambridge, UK), Bcl-2 ( 1:1000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and P-gp (1:100, Invitrogen, Milan, Italy). The hybridization was visualized using an enhanced chemiluminescence detection kit (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific Life Technologies Italia, Monza, Italy) and the Versa DOC imaging system (BioRad Laboratories, Milan, Italy). Immunoblots were quantified by densitometry and results were expressed as arbitrary units (protein/GADPH).

For Western blot analyses, polyclonal rabbit antibody against XIAP was obtained from Cell Signaling Technology Inc., Danvers, MA, polyclonal rabbit antibody against survivin from Abcam Limited, Cambridge, UK; mouse monoclonal antibodies against Bcl-2 were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; monoclonal mouse antibody against GAPDH was obtained from Sigma-Aldrich Srl, Milan, Italy. Whole cellular lysates from HL-60 and HL-60 R cells were obtained using RIPA buffer (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and 15 µg of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis. Proteins were electrophoresed onto nitrocellulose membrane (Amersham, Pharmacia Biotech, Milan, Italy) using a semi-dry fast blot apparatus (Bio-Rad, Milan, Italy). Membranes were blocked with 5% (w/v) BSA in PBS-0.1% (v/v) Tween 20 for 1 h and then probed with the anti-XIAP (1:500), anti-survivin (1:2000), anti-Bcl-2 (1:1000), and anti-GAPDH (1:25000) antibodies. Hybridization was visualized using an enhanced chemiluminescence detection kit (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific Life Technologies Italia, Monza, Italy) and the Versa DOC imaging system (BioRad Laboratories, Milan, Italy). Immunoblots were quantified by densitometry and results were expressed as arbitrary units (protein/GADPH).