Rabbit anti total akt antibody

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.

Larval fat body tissues were homogenized in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris–HCl, pH 7.4) using a Tissuelyser-24 grinder (Jingxin, Shanghai, China). After centrifugation at 15,000 g at 4 °C for 20 min, the supernatants were subjected to separation by SDS-PAGE before immunoblotting analysis. The primary antibodies used are rabbit anti-phospho-Akt (Ser473) antibody (1:1000, Cell Signaling, Catalogue no.9271s), rabbit anti-total-Akt antibody (1:1000, Cell Signaling, Catalogue no.9272), mouse anti-mCherry (1:3000, Abbkine, Catalogue no. A02080), mouse anti-α-tubulin antibody (1:4000; Sigma, Catalogue no. T6199). Anti-secondary antibodies are anti-rabbit IgG (1:2000, Cell Signaling, Catalogue no. 5151) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:2000, Cell Signaling, Catalogue no. 7076). Non-saturated bands were quantified on FIJI-ImageJ (National Institutes of Health) and presented as a ratio in relation to total-Akt. At least three biological replicates were quantified.