Molecular Signaling Pathway Analysis of 6-Shogaol Treatment

A total of 50,000 cells/well were seeded in a 24-well plate. The cells were treated for 24 h with 10–50 µM 6-shogaol concentrations at 37 °C and 5% CO₂. After the treatments were complete, cells were fixed with 1.6% formaldehyde for 10 min. Subsequently, cells were permeabilized with methanol for 1 h. The treatments were replaced with two changes of 600 µL staining medium (1× PBS and 2% BSA) and then incubated with a different antibody. The following human antibodies were from Cell Signaling: anti-mTOR (#2972), anti-phospho-mTOR-S2448 (#2971), anti-PTEN (#9552), anti-phospho-PTEN-S380/T382/383 (#9554), anti-Akt (#9272), anti-phospho-Akt-Ser473 (#9271), anti-GAPDH (14C10), and rabbit mAB (#2118) (Danvers, MA, USA). Anti-β-actin was from ABCAM reference (#ab8227) (Cambridge, MA, USA) and was used as the loading control. The secondary antibodies used were either anti-mouse Alexa 488 (1:400) and anti-rabbit Alexa 488 (1:400). Mean intensity fluorescence was measured with a green laser (430 nm) while using Flow Cytometer Guava easyCyte (Merck Millipore, Darmstadt, Germany). Incubations with only secondary antibody were used as the negative controls [47 (link)].