Anti telomerase

Verification of the extracted exosomes was determined by Western blotting using antibodies against well-characterized exosomal protein markers: CD63, CD9 and TSG101. Extracted exosomes were resuspended in RIPA buffer, sonicated and quantified using Pierce BCA Protein Assay Kit (Thermo Scientific, MA, USA). 50μg of protein was subjected to 10% Sodium Dodecyl Sulfate Poly Acrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was then hybridized for 16h at 4°C with antibodies against exosomal markers: anti-CD63, anti-CD9 (1:1000, Santa Cruz Biotech, TX, USA) and anti-TSG101 (1:500, Abcam MA, USA). In the following day the membrane was subjected to fluorescent labeled secondary antibodies. Visualization was done by the Odyssey analysis software (Odyssey IR imaging system; LI-COR).
The levels of telomerase in pHFF cell line were evaluated by Western blotting as well. The cells were grown for 72hr in the presence of Jurkat derived exosomes. 50 μg of protein were separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and hybridized for 16h at 4°C with specific antibodies: anti-telomerase (1:500, Abcam, MA, USA) Signals were visualized after exposing the membranes to 2^nd fluorescent antibodies and quantified by the Odyssey analysis software (Odyssey IR imaging system; LI-COR).

For western blot analysis, polypeptides in whole cell lysates were resolved by SDS-PAGE and transferred to PVDF membrane filters. Proteins were detected with a 1:1000 or 1:5000 dilution of primary antibody using an enhanced chemiluminescence (ECL) system. Images were acquired using the LAS4000 system (GE Healthcare, Uppsala, Sweden) and Chemidoc-it 410 imaging system (UVP, Upland, CA, USA). The following primary antibodies were used: anti-telomerase (Abcam, Cambridge, UK, ab32020), anti-AMPKα1 (Cell Signaling Technology, Beverly, MA, USA, #2532), anti-acetyl-CoA carboxylase (ACC) (Cell Signaling Technology, #3676), anti-phospho acetyl-CoA carboxylase (p-ACC) (Cell Signaling Technology, #3661), anti-c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc788) and anti-actin (ABM, Richmond, BC, Canada, G043).