Phosphorylated pi3k

MBECs (5x10⁶) were lysed in RIPA buffer (M-PER reagent for the cells and T-PER reagent for the tissues; Thermo Fisher Scientific, Inc.). Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Protein (20 µg) was electrophoresed on a 15% SDS-PAGE gel and transferred to a PVDF membrane (EMD Millipore). The membranes were blocked using 5% BSA at 4˚C overnight. The primary rabbit anti-rat antibodies used in the immunoblotting assays were as follows: PI3K (1:1,000; cat. no. ab76315; Abcam), phosphorylated PI3K (1:500; cat. no. ab182651; Abcam), AKT (1:500; cat. no. ab185633; Abcam), phosphorylated AKT (1:1,000; cat. no. ab133458; Abcam) and β-actin (1:2,000; cat. no. ab8226; Abcam). After incubation, the membrane was washed three times in PBST and incubated with HRP-conjugated goat anti-rabbit IgG mAb (1:2,000; cat. no. PV-6001; OriGene Technologies, Inc.) for 1 h at 37˚C. After washing three times with PBST, the membrane was developed using a chemiluminescence assay system (EMD Millipore). Densitometric quantification of the immunoblot data was performed using Quantity-One 1.2 software (Bio-Rad Laboratories, Inc.).