Quantiscan

Signal intensities of the digitized images were analyzed using a combination of densitometry and volumetry as described in detail before (Wicht et al., 1999 (link)) either using the inbuild software of the luminescence readers or using the program QuantiScan by Biosoft (Oxford, United Kingdom). Each area/density value for a specific protein band was normalized against the corresponding ß-actin or GAPDH signal of each extract. A detailed description of the (semi)quantification procedure is provided in the supplements (Supplementary Figure S9).
Two group comparisons with groups larger than two were performed using one-way ANOVA, followed by the Bonferroni post hoc test. The criterion of significance was P < 0.05, with analysis performed using GraphPad Prism 5.1d (GraphPad, San Diego, CA, USA).

C2C12 myotubes were seeded (1.5 × 10⁵ cells) in 6-well microtiter plates. After stable attachment, cells were preincubated with HCF1/BSS-containing medium for 8 h. The preincubated cells were then lysed with 300 μL of sodium dodecyl sulfate- (SDS-) lysis buffer (20 mM Tris-Cl, 2 mM ethylenediaminetetraacetic acid (EDTA), 3 mM ethylene glycol tetraacetic acid (EGTA), 1% (w/v) Triton X-100, 10% (w/v) glycerol, 5% SDS, 1 mM dithiothreitol (DTT), protease inhibitor cocktail, and phosphatase inhibitor cocktail 3, pH 7.5). The lysate was further centrifuged at 12000 ×g at 4°C for 10 min to remove cell debris. The extent of AMPK activation was assessed from the relative ratio of phospho-AMPKα (p-AMPK) and AMPKα by Western blot analysis using anti-p-AMPKα1/2 antibody and anti-AMPKα1/2 antibody following SDS-PAGE analysis, with a separating gel of 10% (v/v) acrylamide. Cell lysate (20 μg) was loaded and β-actin was used as a reference marker. The immune-stained protein bands were analyzed by densitometry (Quantiscan, Biosoft), and the amounts (arbitrary units) of p-AMPKα and AMPKα were normalized with reference to the β-actin level (arbitrary units) in the sample.