AMPK Methods and Protocols

12. Blotting paper.


13. Tris-buffered saline (TBS): 50 mM Tris–HCl (pH 8),
    150 mM NaCl.


14. TBS-T: TBS, 0.1% (v/v) Tween 20.


15. Blocking solution/antibody diluent solution: TBS-T, 5%
    (w/v) BSA.


16. Stirring plate.


17. ECL substrate solution (e.g., BM chemiluminescence blotting
    substrate POD, SIGMA). Prepare 30 ml of luminescence sub-
    strate solution A with 300μl of starting solution B.


18. Films: FUJI Super RX 18x24 500 E.


19. Developer buffer: Kodak®X-Ray GBX.


20. Fixator buffer: Kodak®X-Ray GBX.


21. ImageJ software (National Institute of Health, Bethesda).
    Antibodies


22. Anti-p70S6K (Cell signaling, 9202): for IB 1:1000 in TBS-T,
    5% (w/v) BSA.


23. Anti-p70S6K Thr389 (Cell signaling, 9234): for IB 1:1000 in
    TBS-T, 5% (w/v) BSA.


24. Anti-eEF2 (Thermo scientific, PA5-17794): for IB 1:1000 in
    TBS-T, 5% (w/v) BSA.


25. Anti-eEF2 Thr56 (Cell signaling, 2331): for IB 1:2000 in
    TBS-T, 5% (w/v) BSA.


26. Anti-GAPDH (Cell signaling, 2118): for IB 1:50,000 in
    TBS-T, 5% (w/v) BSA.


27. Goat anti-rabbit IgG HRP-conjugated (Sigma, A0545): for IB
    1:20,000 in TBS-T, 5% (w/v) BSA.

2.4 Evaluation
of Cardiomyocyte
Hypertrophy by
α-Actinin
Immunostaining

Cell plating

1. Microscope cover glasses, 12 mm diameter.


2. Gelatin: 0.2% gelatin in sterile water (pre-warmed at 37C).


3. Cell culture dishes: 3 cm diameter (9.6 cm^2 ).


4. Ethanol.


5. 1PBS.
   Cell treatments


6. PE, phenformin and AICAr (as described in Subheading2.2,
   items 3– 5 ).


7. Full IMDM medium (as described in Subheading2.2,item 6).


8. IMDM medium without serum (as described in Subheading
   2.2,item 7).

326 Florence Mailleux et al.