Genegnome hr imaging system

MH-S were seeded on 6-well tissue culture plates at 2 × 10⁶ cells/well. Cells were infected as described previously. At indicated time points, cells were washed 3 times with cold PBS, scraped and lysed with 100 μl lysis buffer (1 × SDS Sample Buffer, 62.5 mM Tris-HCl pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue) on ice. Samples were sonicated, boiled at 100°C for 10 min and cooled on ice before polyacrylamide gel electrophoresis and Western Blotting.
Total Akt and Akt and AS160 phosphorylation (P-Akt, P-AS160) were detected with primary rabbit anti Akt (Akt1, Akt2 and Akt3), primary rabbit anti-phospho Ser473 Akt and primary rabbit anti-phospho Thr642 AS160 antibodies (Cell Signaling Technology) diluted 1:1.000 and with secondary goat anti-rabbit antibody conjugated to horseradish peroxidase (Thermo Scientific) diluted 1:10,000. Tubulin was detected with primary mouse anti-tubulin antibody (Sigma) diluted 1:3000 and secondary goat anti-mouse antibody (Pierce) conjugated to horseradish peroxidase diluted 1:1,000. To detect tubulin, membranes were reprobed after stripping of previously used antibodies using Western Blot Stripping Buffer (Thermo Scientific). Images were recorded with a GeneGnome HR imaging system (Syngene).