Polyclonal anti mtor and p70s6k

Small pieces of fresh xenografts were immediately homogenized in protein lysis buffer and centrifugated at 12,000 rpm for 20 minutes, and then the supernatant was harvested as the total cellular protein extracts. The protein concentrations were determined using Bradford method [24 (link)]. Equivalent amounts of proteins (50 μg) were separated by SDS-PAGE and electrotransferred to supported nitrocellulose membranes (Amersham, Sweden) by a semidry transferor. After the membranes were blocked for 2 hours in blocking buffer (5% skimmed milk in PBS-T containing 0.05% Tween 20) at RT, they were incubated with the different primary antibodies: rabbit polyclonal anti-mTOR and p70S6K, mouse monoclonal anti-4EBP1 and p-4EBP1 of 1 : 200 and β-actin of 1 : 400 (Santa Cruz Biotechnology, USA), and mouse monoclonal anti-p-p70S6K of 1 : 2,000 (Cell Signaling Technology, USA) diluted in 1% skimmed milk in PBS-T, respectively, at RT for 2 hours, followed by being incubated with the appropriate HRP-linked secondary antibodies. Finally, the bands of specific proteins on the membranes were visualized with chemiluminescent substrate (Santa Cruz Biotechnology, USA) according to the manufacturer's instructions. The membranes were rinsed three times with PBS-T between the incubations described above [18 (link)].

Small pieces of fresh xenografts were immediately homogenized in protein lysis buffer and centrifugated at 12,000 rpm for 20 minutes, and then the supernatant was harvested as the total cellular protein extracts. The protein concentrations were determined using Bradford method [24 (link)]. Equivalent amounts of proteins (50 μg) were separated by SDS-PAGE and electrotransferred to supported nitrocellulose membranes (Amersham, Sweden) by a semidry transferor. After the membranes were blocked for 2 hours in blocking buffer (5% skimmed milk in PBS-T containing 0.05% Tween 20) at RT, they were incubated with the different primary antibodies: rabbit polyclonal anti-mTOR and p70S6K, mouse monoclonal anti-4EBP1 and p-4EBP1 of 1 : 200 and β-actin of 1 : 400 (Santa Cruz Biotechnology, USA), and mouse monoclonal anti-p-p70S6K of 1 : 2,000 (Cell Signaling Technology, USA) diluted in 1% skimmed milk in PBS-T, respectively, at RT for 2 hours, followed by being incubated with the appropriate HRP-linked secondary antibodies. Finally, the bands of specific proteins on the membranes were visualized with chemiluminescent substrate (Santa Cruz Biotechnology, USA) according to the manufacturer's instructions. The membranes were rinsed three times with PBS-T between the incubations described above [18 (link)].