Western Blot Analysis of Cell Signaling Proteins

Western blot analysis was performed essentially as described [19 (link)]. The method used to normalize the protein levels was “Pierce BCA Protein Assay Kit” (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates (15 μg total protein per lane) were prepared in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-PAGE, and blotted on polyvinylidene difluoride membranes. The following antibodies were used: mouse anti-human β-actin (Proteintech, IL, USA), perilipin (Cell Signaling, MA, USA), phosphor-P70S6K (Cell Signaling, MA, USA), total P70S6K (Cell Signaling, MA, USA), pERK1/2 (Cell Signaling, MA, USA), total ERK1/2 (Cell Signaling, MA, USA), anti-mouse IgG HRP conjugate (Proteintech, IL, USA), and rabbit anti-rat IgG HRP (Proteintech, IL, USA). Image J software was used for densitometric analyses.