Figure 7
Skin A4.74-antigen is distinct from fast skeletal myosin IIA and co-purifies with keratin. A. Western analysis of P9 rat tail skin proteins (Sk) shows that proteins around Mr 230000 are detected by A4.74, but not by F1.652 or A4.1025. A4.74 detects a protein of Mr 220000 typical of skeletal muscle MyHC fast IIA in samples of P29 rat extensor digitorum longus (edl) muscle (Mu) but no proteins in P7 liver (Li). A similar Mr 220000 band was detected with anti-embryonic skeletal myosin antibody F1.652 in neonatal P7 lower hindlimb muscle (Mun) and with the general anti-sarcomeric myosin antibody A4.1025 in P35 lower hindlimb muscle (Mua). The faint Mr~45 000 band (arrowhead) in the skin sample is a non-specific cross-reaction, as the control secondary antibody alone detects this band (2°). Comparable total protein (~50 μg) was loaded per lane and checked by Coomassie stain of a duplicate gel. B. The A4.74-antigen in skin (Sk) was more readily detected than fast IIA MyHC in P29 edl (Mu) muscle in Western blots, even when more muscle protein was loaded as indicated with Coomassie blue staining in identical lanes. C. When skin is dissociated in 6 M urea, the A4.74-reactive proteins (upper panel) are fully dissolved and isolated quantitatively in a high speed supernatant (prd) which contains a wide range of proteins including abundant keratins (bands below Mr 66000, lower panel, Coomassie blue stain of a replicate gel). In this experiment, absolute loaded protein was not determined but a precisely similar proportion of the total sample was run in each lane, as can be seen from the Coomassie stain. After dialysis into PBS to remove urea, no protein or antigenic reactivity is lost (pd). Upon re-centrifugation, all detectable A4.74-reactive protein is found in the pellet (p) associated with keratins, whereas many other proteins remain in the supernatant (s/n). Control antibody F1.652 demonstrates the non-specificity of the low Mr band (arrowhead), but shows no reactivity to A4.74-antigen, whereas A4.74 detects the Mr 220000 skeletal IIA fast MyHC in P29 edl muscle extract (Mu). Each skin lane contains an equivalent proportion of the original sample.