Wga agarose

Frozen muscle samples and fresh skin fibroblasts were homogenized in RIPA buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% IGEPAL, 0.1% SDS) containing protease inhibitor mixture. Western blots of glycoproteins were enriched with wheat‐germ agglutinin (WGA) agarose (Sigma‐Aldrich) as described previously (Michele et al, 2002). Equivalent amounts of protein lysates non‐incubated or incubated with WGA agarose beads were resolved on 7–10% SDS–PAGE gels and transferred to PDVF membranes (Millipore). Immunoreactivity was detected with secondary antibodies conjugated to horseradish peroxidase (Jackson Immuno Research) and developed with SuperSignal West Femto (Thermo Scientific) using an ImageQuant LAS 4000 MiniGold System (GE Healthcare).

Western blots were performed as described previously [20 (link),24 (link)]. Recombinant proteins or cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA, USA). After blocking with Blocking One solution (Nacalai Tesque, Tokyo, Japan), the membranes were incubated with primary antibodies, followed by incubation with respective horseradish peroxidase (HRP)-conjugated secondary antibodies. For the detection of α-DG, the lysates were enriched by wheat germ agglutinin (WGA)-agarose (Sigma-Aldrich) and subjected to Western blot analysis. The protein bands were developed with Immobilon Western Chemiluminescent HRP substrate solution (Millipore) and were imaged with an Amersham™ Imager 600 (GE Healthcare, Chicago, IL, USA). After removing the antibodies or avidin by Western Blot Stripping Buffer (Takara Bio Inc.), the membrane was reproved using different antibodies.