Rabbit polyclonal anti akt 9272

CMT-U27 cells were homogenized in cold lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich). Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked by 5% skim milk and incubated with the following primary antibodies in a blocking buffer overnight at 4ºC: Rabbit polyclonal anti-AKT (9272; Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit polyclonal anti-p-AKT (9271; Cell Signaling), rabbit monoclonal anti-mTOR (2983; Cell Signaling), rabbit monoclonal anti-p-mTOR (5536; Cell Signaling), Rabbit polyclonal anti-p-PI3K (AF3242; Affinity biosciences, Cincinnati, OH, USA), rabbit polyclonal anti-PTEN (bs0686-R; Bioss, Inc., Beijing, China), mouse monoclonal anti-p-PTEN (sc-377573; Santacruz Biotechnology, CA, USA), and mouse monoclonal anti-β-actin (A5441; Sigma-Aldrich). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. The chemiluminescent signals were enhanced using an HRP substrate (Millipore) and detected using a Fusion FX7 acquisition system (Vilbert Lourmat, Eberhardzell, Germany). The density of each band was measured using the software Quantity One (version 4.6.6) and normalized to β-actin. The relative intensity was expressed as compared to the control.

At 18 h after transfection, cells were rinsed twice with PBS and then
incubated with PBS containing
1 mg ml⁻¹Sulfo-NHS-SS-Biotin (Perbio)
for 30 min at room temperature and then rinsed once with PBS and
twice with PBS containing 200 mM glycine. Cells were then harvested and lysed in PBS, 1%
Igepal and protease inhibitors for 30 min on ice. The detergent
lysates were then clarified by centrifugation (14,000 × g,
30 min, 4 °C). Biotinylated proteins were
precipitated by adding 100 μl of streptavidin-agarose beads
(Perbio) and incubated overnight at 4 °C. The
streptavidin-agarose beads were washed three times and incubated for
1 h at 37 °C with 100 mM dithiothreitol and 2x Laemmli sample
buffer. Eluted proteins were then resolved by SDS–PAGE. The following
Abs were used: rabbit anti-Ca_V2.2 (1:500)52 (link) and rabbit
polyclonal anti-Akt (9272, 1:1,000, Cell Signalling Technology).