Rabbit anti s6k

HEK293 cells were seeded in six-well plates at 40 to 50% confluence and cultured overnight to reach ~70% confluence. Rapamycin was diluted to final concentrations of 0.3 and 30 μM in complete culture medium [Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum] and warmed to 37°C before being added to the cells. At desired times (1 to 60 min) after the addition of rapamycin, the medium was aspirated, and 200 μl of 1× SDS sampling buffer [50 mM tris-HCl, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue (pH 6.8)] was immediately added to cause cell lysis. Cell lysates were then collected into 1.5-ml Eppendorf tubes and sonicated for 15 s before boiling at 99°C for 5 min. Lysates were resolved in 7.5% tris-glycine SDS–polyacrylamide gel electrophoresis gel and analyzed using mouse anti– phospho-S6K (Thr³⁸⁹) (1:1000; Cell Signaling Technology, catalog no. 9206) and rabbit anti-S6K (1:1000; Cell Signaling Technology, catalog no. 9202) primary antibodies. DyLight 800 goat anti-mouse (1:5000; Invitrogen, catalog no. SA5-10176) and DyLight 680 goat anti-rabbit (1:5000; Invitrogen, catalog no. 21109) secondary antibodies were used to reveal signals, and blots were scanned with fluorescent immunoblot instruments and software by LI-COR Odyssey software images.