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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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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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L. J Cancer Res Clin Oncol 2011, 137(5):897?05. 29. Ellis MJ, Tao Y, Luo J, A'Hern R, Evans DB, Bhatnagar AS, Chaudri Ross HA, von Kameke A, Miller WR, Smith I, et al: Outcome prediction for estrogen receptor-positive breast cancer based on postneoadjuvant endocrine therapy tumor characteristics. J Natl Cancer Inst 2008, 100(19):1380?388. 30. Dowsett M, Smith IE, Ebbs SR, Dixon JM, Skene A, A'Hern
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MM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high-salt buffer (same as the low-salt buffer but with 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1 NP-40, 1 deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and Tris-EDTA (pH 8.0), and then eluted with elution buffer (1 SDS, 100 mM NaHCO3). The cross-linking of protein-DNA complexes was reversed by incubation with 5 M NaCl at 65?C for 4 h, and DNA w
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MM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high-salt buffer (same as the low-salt buffer but with 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1 NP-40, 1 deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and Tris-EDTA (pH 8.0), and then eluted with elution buffer (1 SDS, 100 mM NaHCO3). The cross-linking of protein-DNA complexes was reversed by incubation with 5 M NaCl at 65?C for 4 h, and DNA w
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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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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 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