Figure 5
Quantification of desmin O-GlcNAcylation and its interplay with phosphorylation. Changes of desmin O-GlcNAcylation level were determined in whole extract by western blot analysis performed after immunoprecipitation of O-GlcNAcylated proteins from untreated myotubes (C, ●), and Ac4-5S-GlcNAc- and Thiamet G-treated myotubes (5S, ▲ and ThG, ■ respectively). (a) Signals were quantified and expressed as ratio of glycosylated protein/protein level (mean ± SEM), and compared to the control condition. The representative signals for whole extract (WE) and the immunoprecipitated proteins (IP) were shown under the histogram. (b) Desmin was detected using western blot following Phos-tag-PAGE after immunoprecipitation of O-GlcNAcylated proteins (left lane) comparing with the whole protein extract from untreated C2C12 myotubes (right lane). NP corresponded to the non-phosphorylated desmin, and P1 and P2 the phosphorylated forms of desmin. The tendency of difference comparing with control is indicated on histogram (n = 15; N = 3 independent cultures). Uncropped images of blots are presented in Supplemental data.
