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ADAMTS-4 in human herniated disc
Appendix
Western blot analysis for ADAMTS-4 protein
The expression level of ADAMTS-4 protein was examined in 8 herniated intervertebral
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disc tissues of two cases each of protrusion, subligamentous extrusion, transligamentous
extrusion, and sequestration types. RA synovial tissues were used as a positive control.
Proteins were extracted from the specimens using lysis buffer containing 50 mM
Tris-HCl (pH 7.6), 10% glycerol, 5 mM magnesium acetate, 0.2 mM ethylenediamine
tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, and 1% sodium dodecylsulfate.
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The concentrations of the extracted proteins were determined by Bradford method
(Bio-Rad, Hercules, CA). The extracted protein (10 µg/lane) was electrophoresed using
15% polyacrylamide gel, and transferred onto nitrocellulose membranes (Atoh, Tokyo,
Japan). After blocking with 5% of non-fat milk, in Tris-buffered saline (TBS) containing
0.05% Tween 20, the membranes were reacted overnight with anti-human ADAMTS-4
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monoclonal antibody (clone 247-3F6) at a concentration of 0.5 µg/ml at 4˚C. They were
washed and incubated with peroxidase-labeled rabbit anti-mouse immunoglobulin G
(IgG) for 1 hour at room temperature. Then, the membranes were incubated with
chemiluminescence Luminol Reagent (Supersignal, Pierce, Rockford, IL) and
immunoreactive bands were visualized by radiography.
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Mouse monoclonal antibody against human ADAMTS4 was developed by using a
synthetic peptide corresponding to the amino acid sequence of part of the spacer domain
(residues 767-778,C-ASETLSGHGPLA for 247-3F6) as an antigen according to the
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ADAMTS-4 in human herniated disc
methods described previously.40 After screening candidate clones by enzyme-linked
immunosorbent assay with synthetic peptide, 247-3F6 was selected. Specific recognition
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of ADAMTS4 by the monoclonal antibody was determined by immunoblotting with
culture media of Cos-7 cells transfected with FLAG-tagged ADAMTS4.41
Isolation of total RNA and RT-PCR of ADAMTS-4
The tissue specimens were kept in liquid nitrogen, and ground into powder with a hand
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mill. The powdery samples were transferred into 1 ml Isogen (Nippon Gene, Toyama,
Japan) and 200 μl chloroform. Following centrifugation (12,000 x g, 15 min, 4˚C), the
aqueous phase was precipitated with an equal volume of 100% ethanol and then
centrifuged (7,500 x g, 5 min, 4˚C). After washing with 70% ethanol, the precipitate was
dried in evaporator and resuspended with diethylpyrocarbonate (DEPC)-treated water.
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Because of shortage of the sample volume, examination of the ADAMTS4 mRNA
expression was possible in four herniated intervertebral disc tissues (each two samples of
subligamentous extrusion and transligamentous extrusion types). Three samples of RA
synovial tissues were used as a control. After DNase (Promega Biotec, Oakland, CA)
treatment of the extracted total RNA, it was reverse transcribed with reverse transcriptase
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(Promega Biotech) and Oligo–dTs (Applied Biosystems, Foster city, CA). The cDNA
was amplified using a cDNA amplifier (MJ Research, Watertown, MA). The
sense-primer was 5’-GCTCTAGAGACACACGCCT CCGATACAGCTTCTTCGTG-3’
and the anti-sense primer was
5’-CGGAATTCTACCCTTGACTTTTCTTTTCCCAAAAAATA-3’ (GenBank NM
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ADAMTS-4 in human herniated disc
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005099). TaqEx DNA polymerase and the substrate for cDNA were mixed, and the PCR
reaction was carried out for 35 cycles under the following conditions: 94˚C for 60
seconds, 60˚C for 60 seconds, and 72˚C for 60 seconds. As internal positive control, the
mRNA expression of glyceraldehyde phosphodehydrogenase (GAPDH) gene was
analyzed. The primer sequences of GAPDH gene were sense primer
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5’-ACCACAGTCGCCATCAC-3’ and anti-sense primer
5’-TCCACCACCCTGTTGCTGTA-3’ (GenBank NM 002046). The PCR product was
subsequently electrophoresed using 2% agarose gel and observed under ultraviolet light
after staining with ethidium bromide. The sequence of the PCR product was analyzed by
the dye terminator method using Big DyeTM Terminator Cycle Sequencing Ready
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Reaction (Applied Biosystems) and automatic sequencer ABI PRISMTM 310 Genetic
Analyzer (Applied Biosystems) and sequence homology was compared with the human
ADAMTS-4 gene (GenBank NM 005099).
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