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Hieved with a magnetic stirrer. Fluorescence of Ca2+ -bound and unbound Fura-2 was measured by rapidly alternating the dual excitation wavelengths between 340 and 380 nm and electronically separating the resultant fluorescence signals at emission wavelength 510 nm. The autofluorescence of each monolayer was subtracted from the fluorescence data. The ratios (R) of the fluorescence at the two wavele
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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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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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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
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Ne whether phospho-c-Jun binding is mediated through CaMKII-dependent PDGFR transactivation pathway, as shown in Figure 7A, LTAinduced phospho-c-Jun binding to the MMP-9 promoterwas significantly inhibited by pretreatment with TG, KN-62, AG1296, SP600125, or TSIIA, analyzed by a ChIP-PCR assay, suggesting that Ca2+/CaMKII-dependent transactivation of PDGFR and JNK is involved in LTA-induced c-Jun/
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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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Oral effects of tamoxifen and the aromatase inhibitor vorozole in postmenopausal patients with primary breast cancer. J Clin Oncol 2002, 20(4):1026?035. 37. Smollich M, Gotte M, Fischgrabe J, Radke I, Kiesel L, Wulfing P: Differential effects of aromatase inhibitors and antiestrogens on estrogen receptor expression in breast cancer cells. Anticancer Res 2009, 29(6):2167?171. 38. Torrisi R, Bagnard
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Were covered with 1 ml of DMEM/F-12 containing 5 M Fura2/AM and incubated at 37 for 45 min. At the end of the period, the cover slips were washed twice with the physiological buffer solution containing (in mM): 125 NaCl, 5 KCl, 1.8 CaCl2, 2 MgCl2, 0.5 NaH2PO4, 5 NaHCO3, 10 HEPES, and 10 glucose, pH 7.4. The cells were incubated in physiological buffer for further 30 min to complete dye de-esterif