Immunoblotting for Apoptosis Markers

Proteins were extracted from cells using RIPA buffer (Nacalai Tesque, Kyoto, Japan) containing cOmplete ™ Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA) and the protein concentration was adjusted to 1 mg/mL. Immunoblotting was performed as previously described [16 (link)]. The primary antibodies used in the experiments were as follows: rabbit anti-PAFR, # bs-14730R-A750 (Bioss. Inc., Woburn, MA, USA), 1:200; rabbit anti-Erk1/2 # 9102 (Cell Signaling Technology, Beverly, MA, USA), 1:1000; rabbit anti-p-Erk1/2 # 4370 (Cell Signaling Technology), 1:1000; rabbit anti-Akt # 4691 (Cell Signaling Technology), 1:1000; rabbit anti-p-Akt #4060 (Cell Signaling Technology), 1:1000; rabbit anti-Caspase-3 #9662 (Cell Signaling Technology), 1:1000; rabbit anti-Cleaved caspase-3 #9661 (Cell Signaling Technology), 1:1000; and mouse anti-α tubulin # sc-5286 (Santa Cruz Biotechnology, Shanghai, China).