Anti s6k1

Lysates were prepared in 1x Dulbecco’s Phosphate Buffer Saline (DPBS—Invitrogen) containing 1% Nonidet P 40 substitue (NP40 –Signa-Aldrish, MO, USA) and protease inhibitor cocktail (Roche Diagnostics, IN, USA). Total protein was determined by Lowry’s method and 25 mg was loaded on a 4–12% gradient SDS–polyacrylamide gel electrophoresis (Invitrogen). Proteins were transferred to 2 μM nitrocellulose membrane using the Trans-blot turbot kit (Bio-Rad) and blotted over-night with anti-mTOR (rabbit polyclonal, abcam), anti-Rictor (rabbit polyclonal, Cell Signaling), anti-Raptor (rabbit polyclonal, Cell Signaling), anti-Atg5 (mouse monoclonal, Cell Signaling), anti-Atg7 (mouse monoclonal, Cell Signaling), anti-pS6K1 (rabbit polyclonal, Abcam), anti-S6K1 (rabbit polyclonal, Abcam), anti-peIF4E (Ser209) (rabbit polyclonal, Cell Signaling), anti-eIF4E (rabbit polyclonal, Cell Signaling), anti-E1/E2 (rabbit polyclonal, gift from Olivier Schwartz laboratory), anti-C (monoclonal antibody, gift from Olivier Schwartz laboratory), anti-GFP (rabbit polyclonal, Cell Signaling) or anti-GAPDH (rabbit polyclonal, Cell Signaling). Secondary HRP-coupled Abs was detected using ECL Plus (Amersham Pharmacia Biotech).

Western blot was performed as previously reported13 . Briefly, whole-cell lysates were prepared and protein concentration was estimated using a BCA Protein Assay kit (Pierce, Rockford, MA, USA). The immune-blots were probed with primary antibodies overnight at 4 °C followed by secondary antibodies for 1 hr. The following primary antibodies were used according to the manufacturer’s protocols: rabbit anti-DDX5, anti-mTOR, anti-p-mTOR (phospho S2448), anti-S6K1, anti-p-S6K1 (phospho T389), mouse anti-β-actin, peroxidase conjugated goat anti-rabbit or anti-mouse IgG (all from Abcam). The blots were visualized using enhanced chemiluminescence detection kit (Thermo). The bands were scanned and quantified by densitometric analysis using Image J software (National Institutes of Health, Bethesda, MD, USA).

The cells were lysed in lysis buffer (Roche, Penzberg, Germany), and equal amounts of protein were separated by SDS-PAGE. The exosome pellets isolated from the same amount of culture medium (10 mL) were lysed in 200 μL of lysis buffer (Roche), and the same amounts of lysate were loaded in each lane of the gels. Immunoblots were probed with antibodies directed against MDK (rabbit monoclonal anti-Midkine, Abcam, Cambridge, MA, USA), P70S6K P-Ser424 (rabbit polyclonal anti-S6 K1, Abcam), or PDK1 P-Ser241 (rabbit polyclonal anti-PDK1, Abcam). CD63 (rabbit monoclonal anti-CD63, Abcam) and TSG101 (rabbit monoclonal anti-TSG101, Abcam) were used as exosomal markers.
The human and mouse AKT Pathway Phosphorylation Array and MAPK Pathway Phosphorylation Array were performed according to the manufacture’s instructions (RayBiotech, Norcross, GA, USA).