Rabbit anti phosphorylated akt ser 473

The cells were washed with PBS twice, and total EPC proteins were harvested by cell lysis buffer (#9803, Cell Signaling Technology Inc., Danvers, MA, USA). Protein extracts were subjected to 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon-P, Merck-Millipore, Darmstadt, Germany). The following antibodies were used for western blot analysis: rabbit anti-PTEN (1 : 1000; #9559, Cell Signaling Technology Inc., Danvers, MA, USA), rabbit anti-phosphorylated Akt (Ser473) (1 : 1000; #9271, Cell Signaling Technology Inc., Danvers, MA, USA), rabbit anti-total Akt antibody (1 : 1000; #9272, Cell Signaling Technology Inc., Danvers, MA, USA), mouse anti-GTPCH I (1 : 1000; sc-271482, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-β-actin (1 : 1000; #4970, Cell Signaling Technology Inc., Danvers, MA, USA). Proteins were visualized with horseradish peroxidase- (HRP-) conjugated anti-rabbit IgG (1 : 2000; #7074, Cell Signaling Technology Inc., Danvers, MA, USA) or HRP-conjugated anti-mouse IgG (1 : 2000; #7076,Cell Signaling Technology Inc., Danvers, MA, USA), followed by use of the ECL chemiluminescence system (#6883, Cell Signaling Technology Inc., Danvers, MA, USA). The intensity of immunoreactive bands was analyzed and expressed as the ratio of PTEN, anti-phosphorylated Akt, Akt, and GTPCH I to β-actin protein in human EPCs.