- CO 2 Incubator.
Cell fixation - 1PBS.
- Paraformaldehyde (PFA): 4% (w/v) PFA, 0.2% (w/v) Triton
X-100.
α-Actinin immunostaining - Microscope slides.
- Dako pen for immunohistochemistry.
- PBS/BSA solutions: PBS, 1% (w/v) BSA and PBS, 0.1%
(w/v) BSA. - Mouse anti-α-actinin antibody (Sigma, A7811): for immunos-
taining 1:750 in 1PBS, 1% (w/v) BSA. - Alexa Fluor 594 donkey anti-mouse antibody (Invitrogen,
A21203): for immunostaining 1:1000 in 1 PBS, 1%
(w/v) BSA. - Vectashield mounting medium with 1.5μg/ml of 4^0 ,6-Diami-
dine-2^0 -phenylindole dihydrochloride (DAPI). - Structured illumination fluorescent microscope.
- AxioVision 4.8 (or similar) software.
2.5 Evaluation
of NFAT
Transcriptional
Activity
Cell infection and treatment- Cultured neonatal cardiomyocytes (700,000 cells per well).
- Full IMDM medium (as described in Subheading2.2,item 6).
- IMDM medium without serum (as described in Subheading
2.2,item 7). - NFAT/luciferase adenoviral construction: Ad5-NFAT-lucifer-
ase reporter (Seven Hills Bioreagents, JMAd-10) [15]. Store at
80 C(seeNote 5).
5.β-Galactosidase adenoviral construction (used as control).
Store at 80 C. - PE, AICAr, and phenformin (as described in Subheading2.2,
items 3– 5 ).
Cell lysis - Cell culture lysis 5reagent (CCLR, Promega, E1500). Dilute
it in H 2 O to make 1lysis buffer. - Cell scraper.
- 1PBS.
NFAT-mediated luciferase activity measurement - Luciferase assay buffer (Promega, E1500).
Study of Cardiac Hypertrophy and Protein Synthesis 327