Evaluation of radioactive L-phenylalanine incorporation into
proteins- Precipitate proteins from supernatants by adding TCA 100% in
order to reach a final concentration of 10%. Allow correct
precipitation by vortexing. - Keep samples on ice for 20 min in order to precipitate proteins.
- Centrifuge 10 min at 9279gat 4C.
- Discard supernatants.
- Add 900μl of cold NaOH 100 mM in order to dissolve the
pellet containing proteins and vortex until the pellet is
completely dissolved. - Add 100μl of 100% TCA and vortex.
- Keep samples on ice for 20 min in order to precipitate proteins.
- Centrifuge for 10 min at 9279gat 4C.
- Discard supernatants.
- Add 500μl of cold 5% TCA and vortex to detach the pellet (see
Note 17). - Centrifuge for 5 min at 9279gat 4C.
- Discard supernatants, add 1 ml of formic acid (seeNotes 18
and 19 ), and vortex. - Prepare vials for scintillation counter with 5 ml of scintillation
liquid (Ultima Gold). - Put 800μl of each protein samples into vials. Shake vials.
- Count vials in a scintillation counter with appropriate counting
program. - Radioactivity measured, corresponding to C^14 -labeledL-phenyl-
alanine incorporated into proteins during all the incubation
period, is then normalized to protein concentration (evaluated
by protein assay) toallow accurate comparison(seeFig. 2a and e).
3.3 Evaluation
of p70S6K and eEF2
Signaling Pathway
Such evaluation is performed on cardiomyocytes cultured and trea-
ted as described in the previous chapter expect that radioactiveL-
phenylalanine was not used (seeNote 20).
Cell harvest
For this step, keep dishes on ice and perform cell lysis as quick
as possible (seeNote 21).- Rinse each dish/well once with cold PBS.
- Add 90μl of cold lysis buffer and scraped the cells.
- Transfer lysates into 1.5 ml Eppendorf reaction tubes.
- Centrifuge lysates for 15 min at 16,100gat 4C and collect
supernatants.
Study of Cardiac Hypertrophy and Protein Synthesis 331