The luciferase activity of miR 26b transfec tants was normalized

The luciferase activity of miR 26b transfec tants was normalized to the mean luciferase activity selleck Tipifarnib of NC transfectants. Immunofluorescent staining for p65 Cells cultured on coverslips in a 48 well plate, were reversely transfected with 50 nM RNA duplexes for 48 hours and remained untreated or treated with 20 ng mL TNF for 10 minutes. Then the cells were fixed with 4% paraformaldehyde and stained with rabbit monoclonal antibody against p65, followed by incubation with HiLyte Fluor 555 conjugated goat anti rabbit IgG and nuclear counterstaining with DAPI. Fluorescent pictures were photographed with Zeiss Axio Imager Z1. RNA extraction and real time quantitative RT PCR Total RNA was extracted from cultured cells using TriPure Isolation Reagent according to the manufacturers instructions.

For real time quantitative RT PCR analysis of mRNA, Inhibitors,Modulators,Libraries 2 ug of total RNA was subjected to DNaseI diges tion at 37 C for 30 mi nutes and then to heat inactivation of DNaseI at 65 C for 10 minutes, followed by reverse transcription using Moloney murine leukemia virus reverse transcriptase. mRNA level was detected using Power SYBR Green PCR Master Mix and Inhibitors,Modulators,Libraries B actin was used as an internal control. The Inhibitors,Modulators,Libraries primers used for qPCR are listed in Additional file 7 Table S1. For qPCR analysis of miRNA, cDNA was synthesized using the Taq man miRNA reverse transcription kit. The expression levels of miR 26b and the reference gene RNU6B were quantified Inhibitors,Modulators,Libraries using the TaqMan Micro RNA Assay Kit. All reactions were performed on a LightCycler 480 and were run in triplicate. The cycle threshold values did not differ by more than 0.

5 among the triplicates. The levels of target genes were normalized to the levels of the internal control genes to permit the calculation of the 2 Ct value. Immunoblotting Cellular proteins were separated in SDS polyacrylamide gels, electrophoretically transferred to polyvinylidene Inhibitors,Modulators,Libraries difluoride membranes, then detected with antibodies. The sources of antibodies were as follows mouse mAb against IB, phospho Ser32Ser36 of IB and B actin rabbit mAb for p65, phospho Ser536 of p65 and TAK1 rabbit polyclonal antibodies for TAB3 and caspase 3. B actin was used as an internal control. All results were reproduced in three independent experiments, and the representative immunoblots are shown.

Apoptosis analysis For cultured cells, 24 hours after transfection, cells were treated with doxorubicin for 48 hours, then applied to morphological examination and detection of caspase 3 activity. For morphological examination, the cells were fixed with 4% PFA, stained with DAPI and those with condensed or fragmented nuclei were considered as apop totic cells. At least selleck products 500 cells were counted for each sample. The activity of caspase 3 was detected by immunoblotting. Activated caspase 3 resulting from the cleavage of the inactive proenzyme form was indicated as 1719 kDa bands below the full length caspase 3 band.

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