supplementary methods data electrophoretic...

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


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


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


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


1 2 3

REFERENCES 4

5

1. Hoh JFY, McGrath PA, White RI. Electrophoretic analysis of multiple 6

forms of myosin in fast-twitch and slow-twitch muscles of the chick. Biochem J 7

1976;157:87-95. 8

2. Reiser PJ, Kline WO. Electrophoretic separation and quantitation of 9

cardiac myosin heavy chain isoforms in eight mammalian species. Am J 10

Physiol 1998;274:H1048-1053. 11

3. Fritz JD, Swartz DR, Greaser ML. Factors affecting polyacrylamide gel 12

electrophoresis and electroblotting of high-molecular-weight myofibrillar 13

proteins. Anal Biochem 1989;180:205-210. 14

4. Blough ER, Rennie ER, Zhang F, Reiser PJ. Enhanced electrophoretic 15

separation and resolution of myosin heavy chains in mammalian and avian 16

skeletal muscles. Anal Biochem 1996;233:31-35. 17

5. Pereira JS, Greaser M, Moss RL. Pulse electrophoresis of muscle 18

myosin heavy chains in sodium dodecyl sulfate-polyacrylamide gels. Anal 19

Biochem 2001;291:229-236. 20

6. Lucas CA, Hoh JFY. Extraocular fast myosin heavy chain expression in 21

the Levator Palpebrae and Retractor Bulbi muscles. Invest Opthalmol Vis Sci 22

1997;38:2817-2825. 23

7. Hoh JFY, Hughes S, Hale PT, Fitzsimons RB. Immunocytochemical 24

and electrophoretic analyses of changes in myosin gene expression in cat 25

limb fast and slow muscles during postnatal development. J Muscle Res Cell 26

Motil 1988;9:30-47. 27


8. Lucas CA, Hoh JF. Distribution of developmental myosin heavy chains 1

in adult rabbit extraocular muscle: identification of a novel embryonic isoform 2

absent in fetal limb. Invest Ophthalmol Vis Sci 2003;44:2450-2456. 3

9. Hamalainen N, Pette D. The histochemical profiles of fast fiber Type-4

IIB, Type-IID, and Type-IIA in skeletal muscles of mouse, rat, and rabbit. J 5

Histochem Cytochem 1993;41:733-743. 6

10. Pedrosa-Domellof F, Soukup T, Thornell LE. Rat muscle spindle 7

immunocytochemistry revisited. Histochemistry 1991;96:327-338. 8

11. Sokoloff AJ, Li H, Burkholder TJ. Limited expression of slow tonic 9

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189. 11

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