Anti p rps6

Human Kelly neuroblastoma cell lines were obtained from the University of California at San Francisco. Cells were grown in RPMI medium with 10% FBS at 37°C. In most experiments, cells were conditioned in 2% FBS for 5 h and replaced with full medium and recombinant human insulin-like growth factor-I (20 ng/ml) (Invitrogen, Waltham, MA, USA) for 1 h before harvesting. Lysates were collected and sonicated. Primary antibodies were as follows: anti-MYCN, anti-phosphorylated Akt, anti-p-rpS6, anti-Akt, anti-rpS6 (Cell Signaling Technology, Danvers, MA, USA), and β-tubulin (Upstate, Buffalo, NY, USA). Immunoblots were developed with horseradish peroxidase-conjugated secondary antibodies (Calbiochem, San Diego, CA, USA) and Enhanced Chemiluminescence Plus reagents (Amersham, Piscataway, NJ, USA).

Ice-cold PBS was used to rinse cells, followed by lysis with lysis buffer (50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 5 mM EDTA, 0,5% Triton X-100) containing PMSF (1 mM), proteinase inhibitors (Roche, Mannheim, Germany, #1697498) and phosphatase inhibitors (10 mM NaPPi, 200 μM NaVO3, 25 mM NaF). Incubation for 15 min on ice was followed by centrifugation of lysates at 16 000 g for 20 min. Bradford Assays (Bio-Rad, Munich, Germany) were used to measure protein concentrations. For electrophoresis, 20-40 μg of protein was separated with 10-15% polyacrylamid gels and blotted onto nitrocellulose membranes (Bio-Rad) by standard procedures. Membranes where washed, incubated over night with primary antibody, washed again and incubated with secondary antibody (1:3000) coupled to horseradish peroxidase (Bio-Rad). Visualization was performed by an enhanced chemiluminescence detection system (GE Healthcare, Munich, Germany). Following primary antibodies were used: anti-beta-Actin (Sigma, Deisenhofen, Clone AC15, A5441), anti-4ebp1 (Cell Signaling, Boston, #9644), anti-p-4ebp1 (Cell Signaling, #2855), anti-rpS6 (Cell Signaling, #2317), anti-p-rpS6 (Cell Signaling, #4858), anti-p27 (DakoCytomation, Clostrup, Clone SX53G8, M 7203). Everolimus was usually used in concentrations of 1 μM for 72 h, leucine (Sigma-Aldrich, St. Louis, MO) for 2 h at concentrations of 10 mM.

Antibodies used in this study include: anti-U2AF1 (ab86305, Abcam), anti-U2AF2 (sc-48804, Santa Cruz Biotechnology), anti-hnRNP A1 (4B10, Abcam), anti-RPS6 (#2317, Cell Signaling), and anti-pRPS6 (#2211, Cell Signaling), anti-TUBULIN (sc-53646, Santa Cruz Biotechnology), anti-SRSF3 (sc-13510, Santa Cruz Biotechnology), anti-EIF4EBP1 (#9452, Cell Signaling), anti-HNRNPC1/C2 (ab10294, Abcam), anti-Flag (F3165, Sigma Aldrich).