Anti pi3kp110α

TUNEL assay kit was obtained from JIANGSU KEYGEN BIOTECH CO.LTD (Nanjing, China). ECL Western blotting detection system was from Vazyme biotech co., ltd (Nanjing, China). The following antibodies were used: anti-p-Ser473-Akt, anti-Akt, anti-PI3Kp110α (Santa, Cruz, CA, USA), anti-β-catenin (BD, San Diego, CA, USA), anti-p-Ser9-GSK3β, anti-GSK3β, anti-caspase 3, anti-cleaved caspase 3, anti-Bcl-2, anti-Bax, anti-β-actin (Bioworld, St. Louis, MN, USA), anti-TH, (Sigma-Aldrich, St. Louis, MO, USA), anti-DAT (Proteintech, Wuhan, China), anti-NeuN(Abcam, Cambridge, UK). Goat anti-rabbit IgG conjugated with horseradish peroxidase and BCA Protein Assay Kit were from Pierce (Rockford, IL, USA). DAB Peroxidase substrate kit was purchased from Vector Laboratories (Burlingame, USA).

Cells were lysed in a radioimmunoprecipitation assay (RIPA) lysis buffer containing the complete EDTA-free protease and phosphatase inhibitor cocktails and 25 units/mL of the pan-nuclease, benzonase. Cells were lysed by vortexing for 1 h at 10 °C and then cleared of cellular debris by centrifugation at 14,000× g rpm for 10 min at 4 °C. The protein concentration of the cleared lysates was determined using the Bradford protein assay reagent (Bio-Rad, Segrate, Italy). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–20% gradient gels and immunoblotted. Primary antibodies used were as follows: Cell Signaling Technologies (Beverly, MA): anti-Akt (Cat #4691; 1:1000), anti-phospho-Ser473 Akt (Cat #4060; 1:1000), anti-PI3Kp110α (Cat #4249; 1:1000), anti-PI3Kp110β (Cat #3011; 1:1000), anti-PI3Kp110γ (Cat #5405; 1:1000), anti-phospho-GSK-3α/β (Cat #9331; 1:1000), and anti-GSK-3α/β (Cat #9315; 1:1000); Santa Cruz Biotechnology (SCBT; Dallas, TX): Anti-PI3Kp110δ (Cat. sc-7176; 1:1000) and anti-β-actin (Cat. sc-1616; 1:1000).