Briefly, collected media of an appro priate volume were prepared with SDS sample buffer was added per 25 ul reaction with MMP 9 or GAPDH primers and TaqMan probes. The MMP 9 and GAPDH selleck catalog without boiling or reduction, and subjected to 0. 1% gelatin 8% SDS PAGE electrophoresis. After electrophor esis, the gels were washed with 2. 5% Triton X 100 and in cubated in a reaction buffer at 37 C for 12 h. The gel was stained with Coomassie brilliant blue R 250 for visualization. RNA preparation and TaqMan quantitative real time PCR Total RNA was isolated from cancer cells using Trizol according to the manufacturers instructions. Quantitative real time PCR analysis was performed using TaqMan one step PCR Master Mix. Total cDNA primers and probes were designed using commercial software.
The oligonucleotide sequences of TaqMan probes and primers were described in Table 1. Quantitative real time PCR assays were conducted in triplicate on a StepOnePlus sequence detection system. Threshold was set above the non template control back ground and within the linear phase of target gene ampli fication to calculate the cycle number at which the transcript was detected. Transfection and MMP 9 promoter driven luciferase assays The HONE 1 cells were seeded at a concentration of 5 x104 cells per well in 6 well cell culture plates. After 24 h of incubation, pGL3 basic and MMP 9 pro moter plasmid were co transfected with a B galactosidase expression vector into cells using Turbofect as previously described. After 12 h of transfection, the cells were treated with vehicle or STE for 24 h.
The cell lysates were harvested and luciferase activity was determined using a luciferase assay kit. The value of the luciferase activity was normalized to transfection efficiency and monitored by B galactosidase expression. Western blot analysis for determining molecular pathway Total cell lysates or nuclear extracts were prepared as pre viously described. The cell lysates were separated in a 10% polyacrylamide gel and transferred onto a nitrocellu lose membrane. The blot was subsequently incubated with 5% non fat milk in Tris buffered saline for 1 h to block non specific binding, and then overnight with polyclonal antibodies against three MAPKs, Src, FAK, and B actin with the specific antibodies for unphosphorylated or phosphorylated forms.
The blots were then incubated AV-951 with horseradish peroxidase goat anti rabbit or anti mouse IgG for 1 h. Signal was detected by using an enhanced chemi luminescence commercial kit. The relative photographic density was quantitated by scanning the photographic negatives on a gel docu mentation and analysis system. Statistical analysis Statistically significant differences were calculated using the Students t test. Significance was set at p 0. 05. The values are the means standard deviation of at least three independent experiments.
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