Protein Extraction and Detection in Cardiac Cells

Whole-cell lysates of mouse LV and NRVMs were prepared in T-PER (Thermo) containing protease and phosphatase inhibitors (Roche). Nuclear fractions of heart 4 days following sham- or TAC procedures were prepared using NE-PER nuclear cytoplasmic extraction reagents according to the manufacturer’s instructions (Thermo). Equal amounts of protein (3–10 μg) were separated on a 4–20% SDS-PAGE gel (Bio-Rad), transferred to a nitrocellulose membrane, and immunoblotted to detect and quantify specific protein bands using an Odyssey scanner (LI-COR version 3)^{74 (link)}. Proteins were detected with a 1000-fold dilution of the following primary antibodies: FoxO1 (#2880, Cell Signaling and ab39670; Abcam); Phospho-FoxO1 (#9464, Cell Signaling), ERK (#4695, Cell Signaling); Phospho-ERK (#4370, Cell Signaling); 10,000-fold dilution of GAPDH (10R-G109a, Fitzgerald) and tubulin (ab6046; Abcam) and 500-dilution of Dio2 antibody (ab77481, Abcam) antibodies, respectively.