Fig. 2. Developmental expression of MURF2 and association with microtubules, myosin and titin. (A) Developmental time course of differentiating myoblasts probed with anti-MURF2 HPC. A single 60 kDa band is expressed upon myogenic differentiation. (B) Microtubule sedimentation assays were performed using protein extracts from undifferentiated (0 hours) and from differentiated C57 cells (48 hours, 120 hours) using nocodazole (n) or taxol (t). MURF2 was detected in the microtubule pellets of differentiated myotubes. (C-D) Localisation of endogenous MURF2 to microtubules in differentiated C57 cells. Double immunofluorescence was performed using anti-MURF2 and anti-tubulin antibodies. After 24 hours of differentiation (C), MURF2 directly colocalised with microtubules as seen in the overlay (yellow). In cells differentiated for 48 hours (D), some MURF2 protein (arrowhead) did not strictly associate with microtubules (green in overlay). (E) MURF2 interacts with sarcomeric myosin and P-zone titin. MURF2 was expressed in transfected HeLa cells and detected by the anti-MURF2-specific antibody HPC (lane 1). The myosin sedimentation assay demonstrates the presence of endogenous MURF2 in myosin preparations (lane 2); addition of transfected MURF2 increases the myosin-associated MURF2 fraction (lane 3). MURF2 associates with titin A164-169 (lane 5) but not with GST alone (lane 6). (Lane 4) Untransfected HeLa cell lysate control on titin A164-169 beads.

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