Figure 2. Differentiation of human mesenchymal stem cells into adipocytes, osteoblasts, and chondrocytes. Passage 2 human MSCs (a) differentiatiated for 18 days and characterized using (b) Oil Red O (adipocyte), (c) Alizarin Red S (osteoblast) and (d) Alcian Blue (chondrocyte).

a b c d

Figure 3. Scheme for metabolic labeling of hMSCs and differentiated cell types with tetraacetylated azido-sugars.

Figure 6. Differentially labeled protein identification: Expanded regions of TAMRA 2-D gels shown in Figure 5 paired with the same gel after post-staining with SYPRO® Ruby protein gel stain. Red circles denote proteins that were identified by MALDI peptide fingerprinting (see Table 1.) and blue circles represent differentially expressed or modified proteins of interest but were not identified by mass spectrometry.

Figure 8. Multiplex Western blot analysis of galectin 1: GalNAz- and GlcNAz-labeled bone extracts were labeled with biotin-alkyne then blotted and detected with streptavidin Qdot 625 conjugate as described in the text. The blots were secondarily incubated with anti-galectin 1 antibody and detected with Western Breeze ECL. The exact overlay of O-linked sugar staining with the specific galectin 1 signal strongly suggests galectin 1 is modified by O-GlcNAc.

Table 1. Identification of GalNAz or GlcNAz labeled protein excised from 2-D gels.