Anti phospho akt ser473

Western blot analysis was performed as described previously [21 (link)]. The primary antibodies used for Western blot analyses included anti-phospho-Src (Tyr418) (Invitrogen, Carlsbad, CA, USA), anti-phospho-FAK (Tyr576) (Invitrogen), anti-FAK (Invitrogen), anti-GAPDH (Invitrogen), anti-phospho-EGFR (Tyr1068) (Cell Signaling, Beverly, MA, USA), anti-phospho-STAT3 (Tyr705) (Cell Signaling), anti-phospho-PI3K (Tyr458) (Cell Signaling), anti-AKT (Cell Signaling), anti-phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling), anti-SAPK/JNK (Cell Signaling), anti-phospho-Paxillin (Tyr118) (Cell Signaling), anti phosphor-p130Cas (Tyr410) (Cell Signaling), anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT3 (Santa Cruz Biotechnology), anti-PI3K (Santa Cruz Biotechnology), anti-phospho-MEK1/2 (Ser218/Ser222) (Santa Cruz Biotechnology), anti-MEK (Santa Cruz Biotechnology), anti-phospho-ERK (Tyr204) (Santa Cruz Biotechnology), anti-ERK2 (Santa Cruz Biotechnology), anti-Paxillin (Santa Cruz Biotechnology), anti-p130 Cas (Santa Cruz Biotechnology), anti-phospho-AKT (Ser473) (Millipore, Billerica, MA, USA)

Oocytes were collected and placed in sample buffer containing 2-mercaptoethanol for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (cat. No. 30566-22; Nacalai Tesque, Kyoto, Japan). Lysates were separated by electrophoresis (cat. No. 4561035; Bio-Rad, Hercules, CA, USA) and the resolved proteins were transferred onto polyvinylidene difluoride membranes (cat. No. 1704156; Bio-Rad). The membranes were incubated with the following antibodies: anti-Pin1 (cat. No. 3722; 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-MPM2 (cat. No. 05-368; 1:500; Millipore, Billerica, MA, USA), anti-phospho-Akt (Ser473) (cat. No. 9271; 1:1000; Cell Signaling Technology), anti-phospho-mTOR (Ser2448) (cat. No. 5536; 1:1000; Cell Signaling Technology), and anti-β-actin (cat. No. ab8227; 1:1000; Abcam, Cambridge, UK) overnight at 4 °C. Anti-MPM2 is a monoclonal antibody for detection of phospho-Ser/Thr-Pro. Antigens were detected using the ECL Prime Western blotting detection reagent (cat. No. RPN2232; GE Healthcare, Little Chalfont, UK) and the Luminescent Image Analyzer, LAS-3000 (Fujifilm Life Science, Tokyo, Japan). Densitometric analysis of the blots was performed by measuring the intensity of each band using the ImageJ software (NIH, Bethesda, MD, USA), and the data were normalized against the relevant β-actin loading controls.