Alpha tubulin t5168

Pre-designed small interfering RNA (siRNA) directed against human PCTAIRE1 (siRNA#1:1472, siRNA#2:1656) and negative scramble control (#1, #2), were purchased from Life Technologies. Antibodies against PCTAIRE1 (rabbit: HPA001366, Sigma), p27 (mouse G173-524: BD, or rabbit C-19: Santa Cruz), alpha-tubulin (T5168, Sigma) and horseradish-peroxidase (HRP)-conjugated secondary antibodies (GE Health Care) were purchased from the indicated sources.

For immunoblotting, cells were lysed in a buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 20 mM glycerophosphate, 1 mM sodium orthovanadate, 5 mM EGTA, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail. Proteins were separated by electrophoresis in 10–12% polyacrylamide gel in the presence of 0.1% SDS, transferred onto a membrane (Immobilon P), and probed with appropriate antibodies. As primary antibodies, we used antibodies to Foxo1 #2880 (Cell Signaling, Danvers, MA, USA), E1A (M73) sc-25 (Santa Cruz), pan-Ras #OP40 (Calbiochem, San Diego, CA, USA), and alpha-tubulin T5168 (Sigma). Anti-mouse and anti-rabbit antibodies conjugated with horseradish peroxidase (Sigma) were used as the secondary antibodies. Visualization of membrane-bound proteins was performed by enhanced chemiluminescence (ECL, Amersham Biosciences, Buckinghamshire, UK).
Every protein of interest was analyzed at least three times on different sets of samples. The band density was evaluated using ImageJ (1.51q-1, Bethesda, MD, USA). Then, density values were scaled to load control and converted to relative units. The figure plots represent the mean values of several experiments; error bars indicate the standard error of the mean (SEM).

Protein lysates were loaded onto 7.5% or 10% sodium dodecyl sulfate polyacrylamide gels and run through electrophoresis using 20 μg of protein per well. A wet transfer was performed onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, California) and then blocked in 5% BSA (Wisent, St-Bruno, Quebec) in Tris-buffered saline tween 20 (TBST) (Bio-Rad, Hercules, California) for 1 h. Membranes were incubated overnight at 4 °C with the following rabbit primary antibodies: monoclonal p-mTOR #5536S, monoclonal mTOR #2983S, monoclonal p-p70S6K #9234S, monoclonal p70S6K #2708S, polyclonal phosphorylated protein kinase B (p-AKT) #9271S, and monoclonal AKT #4691S (Cell Signaling Technology, Danvers, Massachusetts) which were all diluted 1:1000 in BSA. The mouse primary monoclonal antibody alpha-tubulin #T5168 (Sigma-Aldrich, Saint-Louis, Missouri) was diluted 1:5000 in BSA and used as a loading control. Secondary rabbit or mouse antibodies conjugated to horseradish peroxidase (Cell Signaling Technology, Danvers, Massachusetts) were used for chemiluminescence imaging by a ChemiDoc MP imaging system (Bio-Rad, Hercules, California).