Anti p pi3k antibody

The cells were washed by cold PBS 3 times; then, 150 μL RIPA lysate (Beyotime Biotechnology, Shanghai, China) was added. The cells were lysed in ice water by ultrasound, and the protein content was determined by the Bradford method. An equal amount of proteins was taken from each group for 10% SDS-PAGE, and the proteins on the gel were transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked at 4°C for 1 h and then incubated at 4°C overnight with the following primary antibodies: anti-RUNX2 antibody (1:500; Abcam, USA), anti-Osterix antibody (1:1000; Abcam, USA), anti-OCN antibody (1:500; Abcam, USA), anti-PI3K antibody (1:1000; Abcam, USA), anti-Akt antibody (1:300; Abcam, USA), anti-p-PI3K antibody (1:500; Abcam, USA), anti-p-Akt antibody (1:100; Abcam, USA), and anti-GAPDH antibody (1:1000; Abcam, USA). After being cleaned twice with TBST, the membranes were incubated at room temperature for 1 h with fluorescein-labeled goat anti-rabbit IgG (ab205718, 1:2000). The membrane was visualized with an ECL detection kit (Millipore, Bedford, MA, USA) using a chemiluminescence imaging system (Millipore).