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SUPPLEMENTARY METHODS DATA 1
2
Electrophoretic and immunoblotting analysis of MyHCs 3
Myosin was extracted from rabbit EOMs (all recti and inferior oblique 4
muscles), tongue, vastus lateralis, atria and ventricles of the heart. Two 5
specific regions of the SR were used: (1) the EO MyHC rich zone around the 6
EPZ in the middle of the proximal half; (2) the slow-tonic MyHC rich zone at 7
the distal end. Myosin was extracted as described previously 1 and denatured 8
in sodium dodecyl sulphate (SDS) sample buffer for SDS-PAGE. 9
High-resolution SDS-PAGE was performed to separate MyHC 10
isoforms, the polyacrylamide gel composition was optimised to separate the 11
slow-tonic MyHC band from α-cardiac and β/slow MyHCs, the resolution of α-12
cardiac and β/slow MyHCs has been previously shown to be influenced by the 13
total acrylamide concentration and glycerol content of the separating gel 2. 14
Large format gels were run in a Hoefer Scientific SE 600 unit (Hoeffer 15
Scientific Instruments, San Francisco, CA). The separating gel was composed 16
of 37.5% glycerol, 10% acrylamide (with acrylamide/bis-acrylamide ratio of 17
200:1), 0.2 M Tris (pH 8.8), 0.1 M glycine and 0.4% SDS. Polymerization was 18
initiated in the separating gel with 0.015% N,N,N’,N’-19
tetramethylethylenediamine (TEMED) and 0.1% ammonium persulphate. The 20
stacking gel was composed of 30% glycerol, 4% acrylamide (with 21
acrylamide/bis-acrylamide ratio of 37.5:1), 70 mM Tris (pH 6.7), 4 mM EDTA, 22
and 0.4% SDS. Polymerization was initiated with 0.0225% TEMED and 0.1% 23
ammonium persulphate. 24
Separate upper and lower running buffers were used. The lower buffer 25
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consisted of 0.05 M Tris, 0.38 M glycine and 0.05 % SDS. The upper buffer 1
consisted of 0.05 M Tris, 0.38 M glycine, 0.1% SDS and 10 mM 2-2
mercaptoethanol, the latter has been shown to improve band resolution 3, 4. 3
The gels were run using pulse electrophoresis that improves significantly the 4
resolution of MyHC isoforms 5, at a constant current of 10mA (per 16cm long 5
gel) using continuous on/off pulse cycles of 20 s each, for up to 75 hours at 6
10-12 oC. The gels were stained with Coomassie Brilliant Blue. MyHC bands 7
were Western blotted and stained immunochemically as previously described 8
6. 9
10
Development and characterization of an antibody against slow-tonic 11
MyHC 12
The polyclonal antibody against rabbit slow-tonic MyHC used in this 13
study was developed using as starting material an antibody raised in sheep 14
against chicken ALD using methods previously described 7. This anti-ALD 15
serum was first cross-absorbed against washed myofibrils from adult and 16
newborn rabbit limb and heart muscles as previously described 7. Western 17
blots of EOM MyHCs after SDS-PAGE using the cross-absorbed anti-ALD 18
revealed that it cross-reacted with EO MyHC (data not shown). As the EPZ of 19
rabbit EOM is rich in EO MyHC 8, the anti-ALD serum was further cross-20
absorbed against SDS-denatured myosin from the EPZ of EOMs. This myosin 21
was first adsorbed onto nitrocellulose membrane at a high concentration; the 22
resulting membrane was incubated with a blocking solution containing 3% 23
bovine serum albumin, 150 mM NaCl, 10 mM Tris (pH 8.0) for 1 hour at room 24
temperature. The membrane was then incubated with anti-ALD serum that 25
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had been cross absorbed with myofibrils overnight at 4oC. Fig. S1 shows that 1
after the second cross-absorption, the anti-ALD antibody stained a 2
subpopulation of rabbit EOM fibers, predominantly those in the orbital layer, 3
but failed to stain rabbit fast 2A, 2X and 2B fibers known to be present in the 4
tibialis anterior (TA) and vastus lateralis (VL) 9, slow fibers in the soleus (SOL) 5
which expresses β/slow MyHC, the atrium of the heart (AT) expressing α-6
cardiac MyHC and newborn vastus lateralis (nb VL) muscle fibers expressing 7
embryonic and neonatal MyHCs. This antibody did react, however, with a 8
population of intrafusal fibers (IF, indicated by an arrow) in the TA known to 9
express slow-tonic MyHC 10, 11. 10
We further characterized the specificity of the cross-absorbed anti-ALD 11
antibody by Western blot analysis of rabbit EOM MyHCs after high-resolution 12
SDS gel electrophoresis using separating gels with 10% total acrylamide and 13
37.5% glycerol, and running gels using pulse electrophoresis. This method 14
resolved six MyHC bands from whole SR extract: 2A/embryonic/neonatal, 2X, 15
2B, EO/slow-tonic, α-cardiac and β/slow in the order of increasing mobility 16
(Fig. S2A). However, using myosin from the EPZ which is rich in EO MyHC, 17
anti-EO reacted strongly with the third fast migrating band in Western blots 18
(Fig. S2B), while the twice cross-absorbed anti-ALD did not react with it (Fig. 19
S2C), but reacted with the third fast migrating band of the myosin from the 20
distal segment of SR, which contains slow-tonic MyHC as well as EO MyHC, 21
as indicated by the weak binding of anti-EO (Fig. S2B). These results indicate 22
that slow-tonic MyHC co-migrated with EO MyHC, and that the twice cross-23
absorbed anti-ALD specifically binds slow-tonic MyHC, and is hereafter 24
referred to as anti-slow-tonic antibody. 25
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1
2
Fig. S1. Immunoperoxidase staining with the twice cross-absorbed 3
polyclonal anti-ALD of sections of the adult rabbit extraocular (EO), fast tibialis 4
anterior (TA), fast vastus lateralis (VL), slow soleus (SOL), cardiac atrium (AT) 5
and the vastus lateralis of a newborn rabbit (nbVL). The labelled intrafusal 6
muscles fibers (IF) in the TA are indicated by an arrow. 7
8
9
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1
2
Fig. S2. (A) Protein-stained high-resolution SDS gels of MyHCs from 3
adult rabbit tongue and vastus lateralis muscles (TON+VL), the endplate zone 4
(EO EPZ) and distal region (EO DIS) of extraocular muscle (superior rectus), 5
and cardiac atrium and ventricle (AT + VEN). (B) Protein stained reference 6
gels of EO EPZ and EO DIS with adjacent Western blots stained with anti-EO 7
(ANTI-EO). (C) Protein stained gels as in (B), with adjacent Western blots 8
stained with the twice cross-absorbed anti-ALD (ANTI-ALD). 9
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1 2 3
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