AMPK Methods and Protocols

The water provides a humid environment for the coverslips when they are being incubated with primary/secondary anti- body, thus ...

cells are not exposed directly to them. In that case, after wash- ing, the conditioned medium can be collected over an hour and ...

kinase activation. Expert Opin Investig Drugs 21:1155–1167 Bess E, Fisslthaler B, Fromel T, Fleming I (2011) Nitric oxide-induc ...

Chapter 21 Studying the Role of AMPK in Cardiac Hypertrophy and Protein Synthesis Florence Mailleux, Christophe Beauloye, Jean-L ...

unrelated to metabolic issues [2, 3, 6]. Historically, the first con- nection between AMPK and protein synthesis has been establ ...

p70S6K via the inhibition of its upstream kinase, the mammalian target of rapamycin (mTOR) in its hetero-multimeric protein com- ...

Collagenase type II digestion Full IMDM medium: Iscove’s modified Dulbecco’s medium (IMDM), 10% (v/v) FBS, 2% (v/v) penicillin/ ...

1phosphate buffer saline (PBS): 138 mM NaCl, 2.7 mM KCl, 1.47 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 ,pH7. Cell scraper. Lysis buffer: ...

Blotting paper. Tris-buffered saline (TBS): 50 mM Tris–HCl (pH 8), 150 mM NaCl. TBS-T: TBS, 0.1% (v/v) Tween 20. Blocking solut ...

CO 2 Incubator. Cell fixation 1PBS. Paraformaldehyde (PFA): 4% (w/v) PFA, 0.2% (w/v) Triton X-100. α-Actinin immunostaining Mi ...

Luciferase assay substrate (Promega, E1500). Prepare the luciferase assay reagent: Add 10 ml of luciferase assay buffer to the ...

Add an aliquot of 8–10 ml collagenase type II solution on the cardiomyocytes, and shake the flask in stirring bath at 37C for ...

Dish coating Add 1 ml of 0.2% gelatin solution in each dish and incubate for 15 min (seeNote 10). Remove the solution and leave ...

Evaluation of radioactive L-phenylalanine incorporation into proteins Precipitate proteins from supernatants by adding TCA 100% ...

Fig. 2AMPK activation inhibits phenylephrine-mediated increase in protein synthesis and underlying signal- ing. (a–d) Neonatal r ...

Protein concentration is measured in supernatants by Lowry method using BIO-RAD protein assay kit in a 96-well plate. 20μg of t ...

Incubate the membrane with primary antibody overnight at 4 C on a rotating device. Then, wash the membrane with TBS-T twice fo ...

1 mM phenformin or 1 mM AICAr (AMPK stimulation), and PEþphenformin or PEþAICAr (simultaneous hypertrophic treatment and AMPK st ...

After removing distillated water on microscope slides, put 7μl of Vectashield mounting medium with DAPI (seeNote 28)in the midd ...

Fill plate wells with 20μl of each sample in duplicates in 96-well plate (seeNote 30). Wash the luminometer injector with disti ...