Luminata classico substrate

Phospho-ERK, total ERK, phospho-AKT S473, total AKT, p-p70S6K T389, total p70S6K, p-S6 S235/236, total S6, p-Rictor T1135, total Rictor, MTOR and PARP1 were measured by immunoblotting using antibodies obtained from Cell Signaling Technology, Inc. (Danvers, MA). Aliquots of cell extracts prepared in lysis buffer (0.5% Triton X-100, 50 mM β-glycerophosphate (pH 7.2), 0.1 mM Na₃VO₄, 2 mM MgCl₂, 1 mM EGTA, 1mM DTT, 0.3 M NaCl, 2 μg/mL leupeptin and 4 μg/mL aprotinin) were submitted SDS-PAGE. After electrophoretic transfer to nitrocellulose, filters were blocked in 3% bovine serum albumin (BSA; Cohn Fraction V, ICN Biomedicals, Inc., Aurora, OH) in Tris-buffered saline with 0.1% Tween 20 (TTBS). The filters were then incubated overnight at 4°C with antibodies, washed three times in TTBS and incubated for one hour at room temperature with alkaline phosphatase coupled goat anti-rabbit antibodies. The filters were developed using Luminata Classico substrate (Millipore Corporation, Billerica, MA) according to the manufacturer’s instructions. When blotting phosphorylated proteins, the filters were stripped and probed for the corresponding total signaling enzyme level or Na/K-ATPase α-subunit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as a loading control.

Cardiac microtissue protein was isolated after six days of culture following the manufacturer’s protocol for TRIzol Reagent (Invitrogen). Once the protein was isolated, the protein pellet was solubilized in a 1% SDS solution in de-ionized water. Proteins were separated by electrophoresis in Novex Tris-Glycine gels (Life technologies) and dry transferred using the iBlot (Life technologies) to a nitrocellulose iBlot Transfer Stack (Life technologies). Membranes were probed for Phospho-p44/42 MAPK (pERK1/2) (Cell Signaling Technology), p44/42 MAPK (ERK1/2) (Cell Signaling Technology), or GAPDH (Millipore) antibodies. Secondary antibodies were peroxidase conjugated (DAKO). Membranes were developed with ECL reagent LuminataClassico Substrate (Millipore).