Western Blot Analysis of Protein Expression

Cells were scraped from culture plates and incubated for 20 min in ice‐cold lysis buffer containing protease inhibitor cocktails. Nuclear and cytoplasmic fractions were prepared as described previously.^[29 (link)
^] Total protein (10 µg) was separated by SDS‐PAGE and transferred to a PVDF membrane (Bio‐Rad). The membrane was incubated with primary antibodies, and the protein was visualized with ECL (HRP) (Millipore). The following antibodies were used for western blot analysis: anti‐Pten (Cell Signaling Technology, 9188), anti‐β‐actin (Immunoway, YM3028), anti‐p‐T308‐Akt (Cell Signaling Technology, 13038), anti‐p‐S473‐Akt (Cell Signaling Technology, 4060), anti‐Akt (Cell Signaling Technology, 4691), anti‐cTnT (Abcam, ab8295), anti‐Dnmt3l (Cell Signaling Technology, 13451), anti‐Dnmt3b (Cell Signaling Technology, 48488), anti‐Dnmt3b (ab122932, Abcam), anti‐Igf1r (Cell Signaling Technology, 3027), anti‐p‐S253‐FoxO3a (Abcam, ab47285), anti‐p‐T32‐FoxO3a (Cell Signaling Technology, 9464), anti‐FoxO3a (Cell Signaling Technology, 2497), anti‐Phospho‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 4370), anti‐ Insulin Receptor β (Abcam, ab69508), anti‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 4695), anti‐β‐Tubulin (Cell Signaling Technology, 2146), and anti‐Lamin A/C (Cell Signaling Technology, 4777).