Fig. 3. Isoforms of myofilin in Drosophila and myosin binding to myofilin. (A) An immunoblot of thoraces from wild-type Drosophila (WT) and mutant flies lacking thick filaments in the IFM (Mhc⁷), and isolated wild-type flight muscle (IFM) was incubated with anti-myofilin antiserum. There are five isoforms of myofilin in muscles of the thorax. Mhc⁷ lacks the 20 kDa isoform, which is the sole isoform in IFM. The strip on the right shows a blot of thoraces from wild-type flies incubated with anti-flightin. (B) Overlay assay of myosin binding to immunoblots of Lethocerus IFM. Lane1 was incubated in myosin antibody (MAC 147); lane 2 was incubated in buffer containing Lethocerus myosin (0.1 mg/ml) and then in myosin antibody. Myosin bound to myofilin and to other proteins (paramyosin, Pm; mini-paramyosin, mPm; and actin). Myofilin (30 kDa) has an anomalously low migration rate on SDS-PAGE gels. Myosin also bound strongly to the two myosin light chains (Mlc1 and Mlc2); there are three variants of Mlc2, which differ in the extent of phosphorylation and have slightly different mobilities. Myosin did not bind to flightin (23 kDa). Mhc, myosin heavy chain.