Cells were washed once and DNA was stained with 50 ug mL propidium iodide solut

Next, 1 20 of the resultant cDNA was used for each PCR reaction in a total volume of 25 ul. Primers designed and キナーゼ 阻害剤 utilized for canine STAT3 are listed in Table 1; the annealing temperature for this reaction was 55 C. Pri mers designed and utilized for canine STAT3 transcrip tional targets VEGF and MMP2 and GAPDH and human VEGF and GAPDH were published previously with annealing temperatures, Primers designed and utilized for human STAT3 and MMP2 are listed in Table 1. An annealing temperature of 60ºC was used for PCR reactions with human primers for STAT3 and MMP2. Primers were designed to span at least one intron to identify and eliminate any potential genomic DNA contamination.<br><br> All PCR products were run on a 2% agarose purchase Lenalidomide gel with ethidium bromide and visualized using the Alpha Imager system, To quantitatively measure the effects of treatment on STAT3 expression, canine OSA cells were trea ted with curcumin or FLLL32 for 4 or 24 hours, and RNA was extracted using TRIzol reagent according to the manufacturers instruc tions. cDNA was made from 1 ug total RNA using the Superscript III kit, Real time quantitative PCR was performed using the Applied Biosystems Ste pOne Plus Real Time PCR System. STAT3 and 18S mRNA were detected using Fast SYBR green PCR mas ter mix according to the manufac turers protocol and primer sets are detailed in Table 2. All reactions were performed in triplicate and included no template controls for each gene. Relative expression was calculated using the comparative threshold cycle method, Experiments were repeated 3 times using samples in triplicate.<br><br> Protein lysates were prepared and quantified, separated by SDS PAGE, and Western blotting was performed using previously described methods on 2 × 106 OSA cells after treatment with either curcumin, FLLL32, or DMSO for 24 hours. The membranes were then incubated overnight with anti p STAT3, anti p ERK1 2, anti PARP, anti VEGF, anti ubiquitin, or anti survivin antibody, The mem branes LY2603618 IC-83 were incubated with appropriate horseradish per oxidase linked secondary antibodies, washed, and exposed to substrate, Blots were stripped, washed, and reprobed for b actin, total STAT3 or total ERK1 2, Immunoprecipitation OSA cells were serum starved for two hours then treated with DMSO, 10 uM curcumin, 10 uM FLLL32, or 10 uM MG132 for 4 hours.<br><br> The volume of DMSO added to the vehicle treated wells was the same as that delivered to the drug treated wells. Cells were col lected and lysate prepared as described previously, STAT3 antibody was added to lysates that had been precleared with Protein A Agarose beads and allowed to incubate overnight at 4 C. Protein A Agarose beads were added to the protein lysate and antibody and incubated 1 hour at 4 C then washed three times in cold lysis buffer. This was spun down and super natant separated by SDS PAGE and transferred to a PVDF membrane, Western blotting using an anti ubiquitin antibody was performed after addition of the appropriate secondary antibody. The membrane was stripped and reprobed for total STAT3 or b actin, Images were scanned and analyzed using Image J, Proteasome Inhibition Assay OSA cells were serum starved for 2 hours then treated with DMSO, 10 uM curcumin, 10 uM FLLL32, or 10 uM MG132 for 4 hours.