Milliplex map akt mtor phosphoprotein kit

POT, DMSO7, GEF1 and TRM4 cell lines were tyripsinied and counted. Following seeding of 300,000 cells per well in six-well plates and incubation in a 37 °C and 5% CO₂ cell culture incubator overnight, three biological replicates of each cell lines were treated with DMSO, 40 nM of gefitinib and 100 nM of trametinib for 1 h. After the incubation under those conditions, cells were tyripsinised and centrifuged at 1500 rpm to generate cell pellets. Cell pellets were lysed using MDS Tris Lysis Buffer (Meso Scale Diagnostics) containing phosphatase inhibitor I (Sigma-Aldrich), phosphatase inhibitor II (Sigma-Aldrich), protease inhibitor (Cell Signalling Technology). Protein content of lysed samples was quantified using BCA assay (Sigma-Aldrich). MILLIPLEX MAP Akt/mTOR phosphoprotein kit, MILLIPLEX MAPK/SAPK signalling kit, MILLIPLEX MAP RTK phosphoprotein kit (48-611MAG, 48-660MAG, HPRTKMAG-01K respectively, MerckMillipore) were combined with the following singleplex magnetic bead sets to produce three multiplex Luminex assays; Total HSP27, GAPDH (46-702MAG, 46-710MAG, 46-623MAG, 46-641MAG, 46-608MAG, 46-667Mag, MerckMilipore). Bio-Plex Pro phosphor-PDGFRb and Akt (Thr308) (171-V50018M, 171-V50002, Bio-Rad) were combined into a triplex assay. Manufacturer’s recommendations were followed. Phosphoprotein levels were measured on the Luminex 200 system utilising xPOTENT c3.1 software.

Cells were grown in 25 cm² tissue culture flasks (Corning Inc.) at 20% FBS until approximately 80% confluent then dosed with one of seven drugs (plus 3 DMSO controls) for 1 hour. Lysate was stored at −80°C until required.
MILLIPLEX MAP Akt/mTOR phosphoprotein kit, MILLIPLEX MAPK/SAPK signaling kit, MILLIPLEX MAP RTK phosphoprotein kit (48–611MAG, 48–660MAG, HPRTKMAG-01K, respectively, Merck-Millipore) were combined with the following single-plex magnetic bead sets to produce three multiplex Luminex assays: phospho-NFkB, phospho-SRC, phospho-STAT3, phospho-STAT5 A/B, total HSP27 and GAPDH (46–702MAG, 46–710MAG, 46–623MAG, 46–641MAG, 46–608MAG, 46–667MAG, MerckMillipore). Bio-Plex Pro phospho-PDGFRa, phospho-PDGFRb and Akt (Thr308; 171-V50017M, 171-V50018M, 171-V50002, Bio-Rad) were combined into a triplex assay. Manufacturer's protocols were followed throughout.