Fig. 1. Muscle contains a 250 kDa F-actin binding protein that is related to supervillin. (A) Two antibodies (-H340, -pepA) against human supervillin (asterisk) recognize a larger protein in muscle cells (line). Immunoblots of human cervical carcinoma (HeLa S3) cells (lane 1), human 50MB-1 myoblasts (lane 2), hamster skeletal muscle (lane 3) and rabbit skeletal muscle (lane 4) were probed with affinity-purified rabbit polyclonal -H340 (lanes 1-3) or -pepA (lane 4) antibodies. Each lane of the 5% polyacrylamide SDS-gel was loaded with 100 µg total protein. Supervillin is a 205 kDa polypeptide in non-muscle cells (lane 1, asterisk). (B) Specificity of the -H340 antibody on immunoblots. Affinity-purified -H340 antibody (0.5 µg/ml) was pre-incubated for 1 hour at 0°C without (–) or with (+) bacterially expressed H340 protein (60 µg/ml) before incubation with blot strips containing rabbit skeletal muscle (100 µg/lane) and visualization by ECL. Staining of both the major 250 kDa polypeptide (line) and a less-prominent 95 kDa band are competed by immunogen. Progressive loss of the larger band with time suggests proteolytic degradation. (C) Specificity of the -H340 antibody in immunofluorescence. Phase images (a,c) and indirect immunofluorescence micrographs (b,d) of proliferating 50MB-1 cells stained with affinity-purified -H340 and secondary antibodies. To show specificity, the -H340 antibody used for C and D was pre-incubated with 20 µg/ml of the H340 immunogen for 1 hour before use. Bar, 5 µm. (D) Direct binding of ³²P-labeled F-actin to the 250 kDa supervillin-like protein from mouse muscle. Immunoprecipitation with rabbit IgG as a negative control (lane 1) and with -H340 (lane 2). The polypeptide specifically immunoprecipitated by -H340 IgG binds both ³²P-labeled F-actin (top panel) and -H340 antibody (lower panel). (E) Co-fractionation of the 250 kDa supervillin-like protein with dystrophin and caveolin-3 in the crude plasma membrane fraction from rabbit skeletal muscle (Ohlendieck et al., 1991). Immunoblots with antibodies against H340, dystrophin and caveolin-3 in a higher-density membrane fraction enriched in T-tubules and sarcoplasmic reticulum (lane 1, Dense) and in the low-density membrane fraction enriched in sarcolemmal membranes (lane 2, Light) are shown.