Anykd mini protean tgx stain free gels

RBC membranes were prepared as described previously1 (link), and analysed using Any kD Mini-PROTEAN TGX Stain-Free gels and the V3 Western Workflow (BioRad Laboratories). Following transfer, PVDF membranes were incubated with rabbit polyclonal anti-SMIM1 followed by horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG (Agrisera, Sweden). Bands were visualised on ChemiDoc Touch (BioRad Laboratories) and analysed using ImageLab software v5.2 (BioRad Laboratories).

Western blot analysis was as described previously [55 (link)]. Briefly, protein concentrations were measured using NanoDrop-8000 (Thermo Scientific, Waltham, Massachusetts, MA, USA). A total of 25 µg of each protein sample was resolved on AnykD Mini Protean TGX stain free gels (Bio-Rad Laboratories, Hercules, California, CA, USA) and transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, California, CA, USA). The Trans-Blot Turbo blotting system (Bio-Rad Laboratories, Hercules, California, CA, USA) was used for transfer. Antibodies used were as follows: APP (Cell Signaling Technology, Danvers, Massachusetts, MA, USA) and GAPDH (Santa Cruz Biotechnology, Dallas, Texas, TX, USA). Horseradish peroxidase conjugated antibodies (Cell Signaling Technology) were used prior to chemiluminescent detection (GE Healthcare, Chicago, Illinois, IL, USA). Results were quantified using Image J.

Using standard procedures, clarified supernatants from homogenized muscle tissues or murine serum were analyzed by SDS-PAGE followed by western blot with the following primary antibodies: anti-myostatin prodomain AF1539 (R&D Systems); anti-mature Myostatin ab124621 (Abcam). Immunoprecipitations with SRK-015, IgG control, or GDF8-C1 antibodies from homogenized clarified muscle lysate and murine serum were carried out using the Thermo Scientific Pierce™ Co-Immunoprecipitation Kit according to the manufacturer’s specifications.
For quantitative fluorescent western blots, samples were loaded onto Any kD Mini-PROTEAN TGX Stain-Free Gels (Bio-Rad) and signal was detected by near-IR imaging (Azure c600, Azure Biosystems) and quantified using the western blot analysis tool in AzureSpot software (Azure Biosystems). Band intensity (number of positive pixels per unit area) from pro and latent Myostatin was measured and normalized to total loaded protein per lane. For all data presented, a minimum of three biological replicates were measured to generate the presented average values, and error bars on all graphs represent standard deviations. Statistical significance was determined by t-test (two-tailed, homoscedastic).

Western blot analysis was performed as described previously (Arnoldussen et al. 2015 (link)). Briefly, concentrations of the extracted protein were measured using NanoDrop-8000 (Thermo Scientific). 100 μg of protein for each sample was resolved on AnykD Mini protean TGX stain free gels (Bio-Rad) and transferred to a PVDF membrane (Transblot Turbo Transfer pack, Bio-Rad). The Trans-Blot Turbo blotting system (Bio-Rad) was used for transfer. Antibodies used were as follows; Gja1 (rabbit connexin 43 polyclonal antibody, Cell Signaling Technology), Gjb1 (mouse anti-connexin-32 monoclonal antibody, Millipore), Gjb2 (goat anti-Gjb2 polyclonal antibody, Abcam), Sodium Potassium ATPase alpha 1 (mouse anti-NaK ATPase α1 monoclonal antibody, Abcam) and α-Tubulin (rabbit monoclonal antibody, Cell Signaling Technology). Horseradish peroxidase HRP-conjugated secondary antibodies against rabbit and mouse (both from Cell Signaling Technology) and against goat (Santa Cruz Biotechnology, INC) were used.