Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform
Figure 3
Immunoblots of rat tissues for full-length utrophin and N-utrophin (4–10% SDS-PAGE). (a) Adult tissues with protein loads of ~45 μg (1x), ~90 μg (2x), and ~90 μg for C. All lanes stem from one gel blotted onto nitrocellulose membrane. After staining with Ponceau-S, the membrane was cut for separate immunostaining (1x and 2x together plus lane C separate). Lanes 1x and 2x were incubated with anti-UT11. Anti-UT11 recognises full-length (~395 kDa) and N-utrophin (~65 kDa). C Lanes present competition by preincubation of the serum with recombinant UT11 antigen (10 μL/mL serum) before loading onto the blot. In addition, actin (~44 kDa) was visualised in brain tissue by mixing anti-actin antibody with anit-UT11. Finally, the differently incubated membrane lanes were precisely joined together for exposure on X-ray films. Full-length utrophin is seen in all tissues and N-utrophin in kidney and heart. Competition with UT11 antigen removes full-length and N-utrophin. The second lower band underneath the full-length utrophin may represent a degradation product which is, however, only partially removed by antigen competition. (b) The same approach was employed with the gel comprising H, M, and C, except that anti-actin antibody was added to all lanes for loading control and as internal molecular size marker. Protein load was ~90 μg protein of heart (H) and muscle (M) from young (8 days postnatal) and adult tissues. N-Utrophin can only be seen as a faint band in young heart tissue (arrow). The band of full-length utrophin is greatly reduced in adult muscle (double arrow) and abolished by competition in all samples labeled with C. For comparison, C6 rat glioma cells stained with anti-UT11 displaying full-length and N-utrophin as well as adult muscle stained with anti-DYS12 (a) and NCL-DYS1 (b) derived from different gels are included.
