Anti akt antibody

HMEC-1 cells were seeded at 3 × 10⁵/well in 6-well plates overnight and infected with GAS as described above. Harvested cells were lysed in lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). After centrifugation, the lysates were boiled in sample buffer for 10 min. Samples were then subjected to SDS-PAGE and proteins transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk in PBS, the membranes were incubated overnight at 4°C with primary antibodies, including anti-p70s6k antibody (cs9202), anti-phospho-p70s6k antibody (Thr389) (cs9205), anti-AKT antibody (cs9272), anti-phospho-AKT antibody (Ser473) (cs9271), anti-ERK (p44/42 MAPK) antibody (cs9102), anti-phospho-ERK antibody (Thr202/Tyr204) (cs9101), anti-β1 integrin antibody (cs4706), and anti-β-actin antibody (AC-74; Sigma-Aldrich). The above-named antibodies were purchased from Cell Signaling Technology, except for anti-β-actin antibody. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell signaling Technology) at RT for 1 h, membranes were soaked in ECL solution (PerkinElmer Life and Analytical Sciences, Inc.) and the images were captured by a luminescence imaging system (LAS-4000; Fujifilm).

Western blotting was performed as described previously [40 (link)]. The anti-NHERF1and anti-pS473AKT antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-AKT antibody was from Sigma; anti-α-Tubulin, anti-HDAC and anti-GAPDH were from Santa Cruz Biotechnology; anti-GFP and anti-His antibodies were from MBL (Tokyo, Japan).