To maximize efficiency and data output from our limited sample, a multiplex cell signaling assay was run on the Luminex® platform as an alternative to Western blotting. The MILLIPLEX® MAP Akt/mTOR Phosphoprotein 11-plex Magnetic Bead Kit (#48–611MAG) was used in conjunction with MILLIPLEX® MAP Phospho ERK/MAPK 1/2 (Thr185/Tyr187) Magnetic Bead MAPmate™ and MILLIPLEX® MAP Total β-Tubulin Magnetic Bead MAPmate to simultaneously detect 12 phosphoproteins [phosphorylated p70S6K (Thr412), IRS1 (Ser636), GSK3α (Ser21), GSK3β (Ser9), Akt (Ser473), PTEN (Ser380), IR (Tyr1162/Tyr1163), IGF1R (Tyr1135/Tyr1136), RPS6 (Ser235/Ser236), TSC2 (Ser939), and mTOR (Ser2448), ERK/MAPK 1/2 (Thr185/Tyr187)] with β-tubulin serving as a loading control. The assay was conducted following manufacturer protocol (EMD Millipore Corporation, Billerica, MA, USA).
