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Lected and disrupted by sonication in lysis buffer (25 mM Tris, pH 7.8, 2 mM EDTA, 1 Triton X-100, and 10 glycerol). After centrifugation, aliquots of the supernatants were tested for luciferase activity using the luciferase assay system. Firefly luciferase activities were standardized for b-galactosidase activity.To detect the in vivo association of nuclear proteins with rat mmp-9 promoter, chr
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Yrophosphate, 1 sodium vanadate, 2.5 EDTA, 2.5 EGTA, 0.05 (w/v) Triton X-100, 0.5 (w/v) SDS, 0.5 (w/v) deoxycholate, 0.5 (w/v) NP-40, 5 mg/ml leupeptin, 5 mg/ml aprotinin, and 1 PMSF. The lysates were centrifuged at 45,000 ?g for 1 h at 4 to yield the whole cell extract. The protein concentration was determined by the BCA reagents according to the instructions of the manufacturer. Samples fro
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Yrophosphate, 1 sodium vanadate, 2.5 EDTA, 2.5 EGTA, 0.05 (w/v) Triton X-100, 0.5 (w/v) SDS, 0.5 (w/v) deoxycholate, 0.5 (w/v) NP-40, 5 mg/ml leupeptin, 5 mg/ml aprotinin, and 1 PMSF. The lysates were centrifuged at 45,000 ?g for 1 h at 4 to yield the whole cell extract. The protein concentration was determined by the BCA reagents according to the instructions of the manufacturer. Samples fro
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Or MMP-9 and 514 bp for b-actin) and direct sequence analysis of the PCR product.Preparation of cell extracts and western blot analysisRBA-1 cells were made quiescent at confluence by incubation in serum-free DMEM/F-12 for 24 h. Growtharrested cells were incubated with LTA at 37 for the indicated times. When inhibitors were used, they were added 1 h prior to the application of LTA. Treatment of R
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On of b-actin, a relatively invariant internal reference RNA, was performed in parallel, and cDNA amounts were standardized to equivalent b-actin mRNA levels. These primer sets specifically recognize only the genes of interest asFor experiments, cells were made quiescent at confluence by incubation in serum-free DMEM/F-12 for 24 h. Growth-arrested RBA-1 were incubated with LTA at 37 for various t
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On of these signalingcascades and transcription factors has been reported to be involved in induction of MMP-9 in rat astrocytes [20,24,25]. Moreover, transactivation of receptor tyrosine kinases such as platelet-derived growth factor receptor (PDGFR) by several stimuli has also been implicated in mediating cellular functions of glial cells [29]. More recently, we have demonstrated that LTA induce
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On of these signalingcascades and transcription factors has been reported to be involved in induction of MMP-9 in rat astrocytes [20,24,25]. Moreover, transactivation of receptor tyrosine kinases such as platelet-derived growth factor receptor (PDGFR) by several stimuli has also been implicated in mediating cellular functions of glial cells [29]. More recently, we have demonstrated that LTA induce
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Collected and analyzed for de novo synthesis and activity of MMPs by gelatin zymography. As shown in Figure 1A, pretreatment with TSIIA (0.110 M) significantly attenuated LTA-induced proMMP9 expression and activity. Moreover, pretreatment with TSIIA (10 M) also markedly inhibited LTA (50 g/ml, 16 h)-induced MMP-9 mRNA expression, determined by RT-PCR (Figure 1B), suggesting that AP-1 is an importa