Westran clear signal

For determination of gastrocnemius phospho-eEF2 (Thr 56) protein levels 2 μg/μl gastrocnemius Western blotting preps were made using 4x Laemmli buffer. Thereafter, 30 μl of prepped samples were loaded onto 12% SDS-polyacrylamide gels and subjected to electrophoresis (200 V @ 75 min). Proteins were then transferred to polyvinylidene difluoride membranes (Whatman^™, Westran^® Clear Signal), and membranes were blocked for 1 h at room temperature with 5% nonfat milk powder.
Rabbit anti-phospho-eEF2 (Thr 56) IgG (1:1,000; Cell Signaling) was incubated with membranes overnight at 4°C in 5% bovine serum albumin (BSA), and the following day membranes were incubated with anti-rabbit or anti-mouse IgG secondary antibodies (1:2,000, Cell Signaling), respectively, at room temperature for 1 h prior to membrane development. Membrane development was performed as described above. As described above, membranes were stained with Coomassie in order to visually ensure between-lane protein loading equality.

In order to examine if MSTN treatment reduced muscle protein synthesis (MPS) rates compared to DM/CTL myotubes, the SUnSET method was employed [40 (link)]. Briefly, RIPA homogenates from 145 mm plates were subjected to 4-20% SDS-polyacrylamide gel electrophoresis using pre-casted gels (C.B.S. Scientific Company, San Diego, CA, USA). Proteins were transferred to polyvinylidene difluoride membranes (Whatman™, Westran® Clear Signal), and membranes were blocked for 1 h at room temperature with 5% nonfat milk powder. Mouse anti-puromycin (1:5,000; Millipore) was then incubated with membranes overnight at 4°C in 5% bovine serum albumin, and the following day membranes were incubated with anti-mouse IgG secondary antibodies (Cell Signaling, Danvers, MA, USA) at room temperature for 1 h. Membranes were then developed using an enhanced chemiluminescent reagent (Amersham, Pittsburgh, PA, USA), and band densitometry was performed through the use of a UVP Imager and associated densitometry software (UVP, LLC, Upland, CA, USA).