Alphaview software

The electrophoretic mobility shift assay (EMSA) was performed according to Ringgaard et al. [74 (link)]. 6 pmoles of the double-stranded oligonucleotide labeled with the fluorescent dye (Cy3 or Cy5) were incubated with increasing quantities of His₆-ParB protein in the presence of 18 pmoles of non-specific ds oligonucleotides as a competitor DNA in binding buffer (10 mM Tris-HCl pH 7.5, 0.5 mM dithiothreitol, 50 mM KCl, 1 mM MgCl₂) [75 (link)] in a total volume of 20 μl. In dissociation experiments the constant amount of 240 pmoles of His₆-ParB was added to 6 pmoles of fluorescently labeled ds oligonucleotides and increasing amounts of unlabeled ds parS2 oligonucleotide (18, 60, 90, 120, 180 pmoles, respectively). After 15 min incubation at 37°C the samples were separated on 5% polyacrylamide gels in 0.5 x Tris-borate-EDTA buffer (TBE) [70 ]. The DNA was visualized using FluorChemQ MultiImageIII ChemiImager and the images were captured using Alpha View software (Alpha Innotech).

Western blot analysis was carried out as previously described [12] (link). For tissue lysates, 40 µg from each sample was loaded. For serum samples, one µL-equivalent serum samples were suspended in NuPAGE LDS sample buffer and heated for 5 min at 90°C. Samples were electrophoresed on 10% BisTris NuPAGE gels (Invitrogen, Carlsbad, CA), transferred to 0.2 µm Protran BA83 nitrocellulose membranes (GE Healthcare, Piscataway, NJ), and probed with the CTRP2 primary antibody and appropriate HRP-conjugated secondary antibody. Bands were visualized with Immobilon Western HRP substrate peroxide solution (Millipore, Billerica, MA), captured with MultiImage III FluorChem® Q (Alpha Innotech, San Leandro, CA), and quantified using Alphaview Software (Alpha Innotech).

The primary antibodies used were anti-GAPDH (Millipore), anti-Carabin (ProSci Inc.), anti-phospho-mitogen-activated protein kinase kinase 1/2 (MEK1/2) (Cell Signaling), anti-MEK1/2 (Cell Signaling), anti-phospho-CaM KinaseII pThr286 (Thermo Scientific), and anti-CaM KinaseII. Subsequently the Western blot membranes were incubated with a horseradish peroxidase (HRP)–coupled secondary antibody (GE Healthcare). Visualization was achieved by applying Supersignal West Pico- (Thermo Scientific) or Immobilon Western- (Millipore) chemiluminescence HRP substrate. Finally, image acquisition and densitometry were performed using Alphaview software (Alpha Innotech).

Diaphragm muscles were prepared in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS) with protease and phosphatase inhibitor cocktails (Sigma). The protein concentration was determined using a Coomassie Plus protein assay reagent (Thermo Scientific). For each sample, 10 µg of protein were loaded and separated on a SDS-polyacrylamide gel, according manufactures direction (BioRad). The proteins were then transferred to Nitrocelluloseous membranes blocked with milk and incubated with Rabbit polyclonal anti-peptide antibody that can recognize myonectin (epitope 77-KQSDKGI NSKRRSKARR-93). Myonectin antibody was kindly provided by the lab of GW Wong and had been used previously (Seldin et al., 2012 (link)). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was purchased from Novus Biologicals (NB300-221), COX IV antibody was purchased from Cell Signaling Technology (4844), and FNDC5 antibody was purchased from Abcam (ab131390). Antibody detection was performed with the appropriate horseradish perioxidase-conjugated secondary antibodies. Visualizations were completed with MultiImage III FuorChem^® M (Alpha Innotech) and quantifications were performed by Alphaview Software (Alpha Innotech).

Protein immunodetection by Western blot (WB) was performed after separating proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TGX Criterion 4–15% gel (Bio-Rad) was used for misfolded rec-PrP samples and 4–12% NuPAGE Midi gel (Invitrogen Life Technologies) for mammalian samples. The recombinant proteins were electroblotted onto polyvinylidene fluoride (PVDF) membranes (Trans-Blot Transfer Pack Turbo PVDF, Bio-Rad) and the mammalian proteins onto nitrocellulose membranes (Protran BA85, GE Healthcare). Membranes were probed with antibody 3F4 (diluted 1:10,000 in 0.1% (w/v) of fat-free powder milk in phosphate buffered saline (PBS, Fisher Bioreagents) with 0.05% Tween-20 (Sigma-Aldrich, Atom) as described previously^{75 (link)}. The immunoreactive bands were visualized by chemiluminescence using the Super Signal West Pico kit (Thermo Scientific Pierce) and the digital images were recorded by FluorChem Q (Alpha Innotech).
Densitometric analysis of the immunoreactive bands obtained by WB was carried out using the AlphaView software (Alpha Innotech). The images were analysed by densitometry to evaluate the relative amount of immunoreactive band signal strength, quantified in terms of optical density and translated to percentages.