Rabbit anti pakt t308

Cells grown on coverslips were fixed with 4% formaldehyde (Polysciences, Inc, Warrington, Pennsylvania) in PBS at RT for 15 min. Coverslips were washed in PBS 3 times for 5 min and blocked in blocking buffer (1xPBS, 5% normal goat serum (Cell Signaling), 0.3% Triton X-100) for 1 hr. Cells were incubated with primary antibodies diluted in 1xPBS, 1% BSA, 0.3% Triton X-100 overnight at 4°C. Followed by rinsing 3x 5 min with PBS, coverslips were incubated with secondary antibodies diluted in 1xPBS, 1% BSA, 0.3% Triton X-100 for 1–2 hr at RT in dark. Cells were then mounted on glass slides using ProLong Gold with DAPI (Cell Signaling) mounting medium and imaged with a Nikon TE2000-U epifluorescence microscope with appropriate filters. Corrected total cell membrane fluorescence was determined for p-Akt T308 and p-Akt S473 signals using Image J (National Institute of Health, Bethesda, Maryland. Rabbit anti–p-Akt T308, anti-p-Akt S473 and mouse anti-HA were all from Cell Signaling. Alexa 488 anti–rabbit, Alexa 594 anti–mouse from Invitrogen. Quantifications are average of 3 independent experiments of at least 10 cells per experimental condition.

Protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 8 % gels) and electrotransferred (semi-dry transfer) to polyvinylidene difluoride membranes (Immobilon-P, Millipore). The membranes were blocked with 10 % (w/v) dried nonfat milk powder in Tris-buffered saline with 0.1 % Tween-20 and incubated with the following primary antibodies: rabbit anti-pGSK-3α/β(Ser21/9) (1:1000 dilution; #9331, Cell Signaling Technology); rabbit anti-GSK-3α/β (1:1000 dilution; #5676P, Cell Signaling Technology); rabbit anti-pAktS473 (1:1000 dilution; #4060, Cell Signaling Technology), rabbit anti-pAktT308 (1:1000 dilution; #4056, Cell Signaling Technology); rabbit total Akt (1:1000 dilution; #9272, Cell Signaling Technology); mouse anti-β-dystroglycan (β-DG; 1:500 dilution; NCL-b-DG, Novocastra); and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:2000 dilution; MAB374, Chemicon). Following washing with TBST, the membranes were incubated with horseradish peroxidase-labeled secondary antibody (anti-mouse or anti-rabbit; Vector Laboratories). After washing, peroxidase activity was visualized with ECL plus reagent (GE Healthcare). Signal densities were analyzed using GeneTools software (SynGene, England).