SUPPLEMENTARY METHODS DATA 1 1 1 1 1 1 1 1 0 1 Electrophoretic and immunoblotting analysis of MyHCs Myosin was extracted from rabbit EOMs (all recti and inferior oblique muscles), tongue, vastus lateralis, atria and ventricles of the heart. Two specific regions of the SR were used: (1) the EO MyHC rich zone around the EPZ in the middle of the proximal half; () the slow-tonic MyHC rich zone at the distal end. Myosin was extracted as described previously 1 and denatured in sodium dodecyl sulphate (SDS) sample buffer for SDS-PAGE. High-resolution SDS-PAGE was performed to separate MyHC isoforms, the polyacrylamide gel composition was optimised to separate the slow-tonic MyHC band from α-cardiac and β/slow MyHCs, the resolution of α- cardiac and β/slow MyHCs has been previously shown to be influenced by the total acrylamide concentration and glycerol content of the separating gel. Large format gels were run in a Hoefer Scientific SE 00 unit (Hoeffer Scientific Instruments, San Francisco, CA). The separating gel was composed of.% glycerol, % acrylamide (with acrylamide/bis-acrylamide ratio of 00:1), 0. M Tris (ph.), 0.1 M glycine and 0.% SDS. Polymerization was initiated in the separating gel with 0.01% N,N,N,N - tetramethylethylenediamine (TEMED) and 0.1% ammonium persulphate. The stacking gel was composed of 0% glycerol, % acrylamide (with acrylamide/bis-acrylamide ratio of.:1), 0 mm Tris (ph.), mm EDTA, and 0.% SDS. Polymerization was initiated with 0.0% TEMED and 0.1% ammonium persulphate. Separate upper and lower running buffers were used. The lower buffer Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs// on 0/0/01
consisted of 0.0 M Tris, 0. M glycine and 0.0 % SDS. The upper buffer consisted of 0.0 M Tris, 0. M glycine, 0.1% SDS and mm - mercaptoethanol, the latter has been shown to improve band resolution,. The gels were run using pulse electrophoresis that improves significantly the resolution of MyHC isoforms, at a constant current of ma (per 1cm long gel) using continuous on/off pulse cycles of 0 s each, for up to hours at -1 o C. The gels were stained with Coomassie Brilliant Blue. MyHC bands were Western blotted and stained immunochemically as previously described. 1 1 1 1 1 1 1 1 0 1 Development and characterization of an antibody against slow-tonic MyHC The polyclonal antibody against rabbit slow-tonic MyHC used in this study was developed using as starting material an antibody raised in sheep against chicken ALD using methods previously described. This anti-ald serum was first cross-absorbed against washed myofibrils from adult and newborn rabbit limb and heart muscles as previously described. Western blots of EOM MyHCs after SDS-PAGE using the cross-absorbed anti-ald revealed that it cross-reacted with EO MyHC (data not shown). As the EPZ of rabbit EOM is rich in EO MyHC, the anti-ald serum was further crossabsorbed against SDS-denatured myosin from the EPZ of EOMs. This myosin was first adsorbed onto nitrocellulose membrane at a high concentration; the resulting membrane was incubated with a blocking solution containing % bovine serum albumin, mm NaCl, mm Tris (ph.0) for 1 hour at room temperature. The membrane was then incubated with anti-ald serum that Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs// on 0/0/01
1 1 1 1 1 1 1 1 0 1 had been cross absorbed with myofibrils overnight at o C. Fig. S1 shows that after the second cross-absorption, the anti-ald antibody stained a subpopulation of rabbit EOM fibers, predominantly those in the orbital layer, but failed to stain rabbit fast A, X and B fibers known to be present in the tibialis anterior (TA) and vastus lateralis (VL), slow fibers in the soleus (SOL) which expresses β/slow MyHC, the atrium of the heart (AT) expressing α- cardiac MyHC and newborn vastus lateralis (nb VL) muscle fibers expressing embryonic and neonatal MyHCs. This antibody did react, however, with a population of intrafusal fibers (IF, indicated by an arrow) in the TA known to express slow-tonic MyHC,. We further characterized the specificity of the cross-absorbed anti-ald antibody by Western blot analysis of rabbit EOM MyHCs after high-resolution SDS gel electrophoresis using separating gels with % total acrylamide and.% glycerol, and running gels using pulse electrophoresis. This method resolved six MyHC bands from whole SR extract: A/embryonic/neonatal, X, B, EO/slow-tonic, α-cardiac and β/slow in the order of increasing mobility (Fig. SA). However, using myosin from the EPZ which is rich in EO MyHC, anti-eo reacted strongly with the third fast migrating band in Western blots (Fig. SB), while the twice cross-absorbed anti-ald did not react with it (Fig. SC), but reacted with the third fast migrating band of the myosin from the distal segment of SR, which contains slow-tonic MyHC as well as EO MyHC, as indicated by the weak binding of anti-eo (Fig. SB). These results indicate that slow-tonic MyHC co-migrated with EO MyHC, and that the twice crossabsorbed anti-ald specifically binds slow-tonic MyHC, and is hereafter referred to as anti-slow-tonic antibody. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs// on 0/0/01
Fig. S1. Immunoperoxidase staining with the twice cross-absorbed polyclonal anti-ald of sections of the adult rabbit extraocular (EO), fast tibialis anterior (TA), fast vastus lateralis (VL), slow soleus (SOL), cardiac atrium (AT) and the vastus lateralis of a newborn rabbit (nbvl). The labelled intrafusal muscles fibers (IF) in the TA are indicated by an arrow. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs// on 0/0/01
Fig. S. (A) Protein-stained high-resolution SDS gels of MyHCs from adult rabbit tongue and vastus lateralis muscles (TON+VL), the endplate zone (EO EPZ) and distal region (EO DIS) of extraocular muscle (superior rectus), and cardiac atrium and ventricle (AT + VEN). (B) Protein stained reference gels of EO EPZ and EO DIS with adjacent Western blots stained with anti-eo (ANTI-EO). (C) Protein stained gels as in (B), with adjacent Western blots stained with the twice cross-absorbed anti-ald (ANTI-ALD). Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs// on 0/0/01
REFERENCES 1 1 1 1 1 1 1 1 0 1 1. Hoh JFY, McGrath PA, White RI. Electrophoretic analysis of multiple forms of myosin in fast-twitch and slow-twitch muscles of the chick. Biochem J 1;1:-.. Reiser PJ, Kline WO. Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms in eight mammalian species. Am J Physiol 1;:H-.. Fritz JD, Swartz DR, Greaser ML. Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins. Anal Biochem 1;:0-.. Blough ER, Rennie ER, Zhang F, Reiser PJ. Enhanced electrophoretic separation and resolution of myosin heavy chains in mammalian and avian skeletal muscles. Anal Biochem 1;:1-.. Pereira JS, Greaser M, Moss RL. Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 001;1:-.. Lucas CA, Hoh JFY. Extraocular fast myosin heavy chain expression in the Levator Palpebrae and Retractor Bulbi muscles. Invest Opthalmol Vis Sci 1;:1-.. Hoh JFY, Hughes S, Hale PT, Fitzsimons RB. Immunocytochemical and electrophoretic analyses of changes in myosin gene expression in cat limb fast and slow muscles during postnatal development. J Muscle Res Cell Motil 1;:0-. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs// on 0/0/01
. Lucas CA, Hoh JF. Distribution of developmental myosin heavy chains in adult rabbit extraocular muscle: identification of a novel embryonic isoform absent in fetal limb. Invest Ophthalmol Vis Sci 00;:0-.. Hamalainen N, Pette D. The histochemical profiles of fast fiber Type- IIB, Type-IID, and Type-IIA in skeletal muscles of mouse, rat, and rabbit. J Histochem Cytochem 1;1:-.. Pedrosa-Domellof F, Soukup T, Thornell LE. Rat muscle spindle immunocytochemistry revisited. Histochemistry ;:-.. Sokoloff AJ, Li H, Burkholder TJ. Limited expression of slow tonic myosin heavy chain in human cranial muscles. Muscle Nerve 00;:1-1. 1 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs// on 0/0/01