Total pi3k

The cells were treated with various concentrations of KRG (160, 320, and 640 μg/mL), montelukast sodium (MON, 10μM; Merck & Co., NJ) and DEX (1μM; Sigma, MO) followed by incubation with CSC (100 μg/mL) for the indicated times. The cells were collected by washing twice with PBS and resuspended in RIPA buffer (Sigma–Aldrich) containing a protease/phosphatase inhibitor cocktail (Sigma–Aldrich). Immunoblotting was performed as previously described [21 (link)]. The primary antibodies used were as follows: total PI3K (1:1000 dilution; Abcam), p-PI3K (1:1000 dilution; Abcam), total AKT (1:1000 dilution; Abcam), p-AKT (1:1000 dilution; Abcam), Bax (1:1000 dilution; Abcam), β-actin (1:2000 dilution; Abcam), VEGF (1:1000 dilution; Novus Biologicals), and cleaved Caspase 3 (1:1000 dilution; Cell Signaling Technology). Relative protein expression was determined using Chemi-Doc (Bio-Rad Laboratories).

Cell proteins were isolated using 400 µl of RIPA buffer (Thermo Fisher Scientific, Inc.). Gels were scanned and quantified by densitometry using the Quantity-One 4.4 software (Bio-Rad, Laboratories, Inc., CA, USA). The protein was quantified by using the Bradford method (Bio-Rad, Laboratories, Inc.). The sample solution containing 50 µg proteins were separated by 10% SDS-PAGE, then transferred into nitrocellulose membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 1% Tween (TBS) for 1 h, then incubated with the following primary antibodies overnight at 4°C: Phosphorylated (p-)PI3K (cat no. ab125633, 1:2,000), total PI3K (cat no. ab127617, 1:2,000), p-AKT (cat no. ab38449, 1:2,000), total Akt (cat no. ab126580; 1:2,000) and GAPDH (cat no. ab8245; 1:2,000) (all from Abcam, Cambridge, UK). The secondary antibody (anti-mouse IgG) conjugated to horseradish peroxidase (cat no. 7076, 1:300; Cell Signaling Technology, Inc., Danvers, MA, USA) was incubated with the membranes for 1 h at 37°C. Protein binds were visualized using an enhanced chemiluminescence reagent chromogenic substrate (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. All experiments were performed in triplicate.