Figure 4

Quantification of the phosphorylated forms of desmin in C2C12 differentiated myotubes consecutively to global O-GlcNAcylation variations. Twenty µg of whole proteins samples (RIPA extracts) or protein samples resulting from differential extractions (DSB: soluble; DIB: insoluble; and SDS-resolubilized: cytoskeleton) were separated using Phos-tag-PAGE, and desmin was detected using western blot. (a,b) Quantification of desmin phosphorylation changes in whole protein extract of untreated (C), Ac₄-5S-GlcNAc- and Thiamet G-treated myotubes (5S, and ThG respectively). The phosphorylated forms of desmin were separated according to the number of phosphate groups (P1: first retarded form of desmin; P2: second retarded form of desmin) comparing with the non-phosphorylated form of desmin (NP); the representative pattern in untreated and treated myotubes was presented on (a) panel. The signals were quantified and expressed as the relative percentage of each form (NP, P1 and P2 forms, comparing with the sum of all forms (NP + P1 + P2, equal to 100%, for each condition). (c) A control sample was dephosphorylated using alkaline phosphatase (AP) prior to Phos-tag-PAGE and detection of desmin was carried out through western blot. (d) Protein samples resulting from differential extractions (DSB: soluble; DIB: insoluble; and SDS-resolubilized: cytoskeleton) were separated using Phos-tag-PAGE, and detection of desmin was carried out through western blot. **p < 0.01: significantly different from control (n = 15; N = 3 independent cultures). Uncropped images of blots are presented in Supplemental data.