Membranes were again washed in TBS T three times and developed. MicroRNA overexpression Cells were seeded into six well plates at 60% confluency 1 day before transfection. Transfection mixtures were pre pared in commercial medium, and cells were kept in antibiotic free medium during transfection. miR 32 overexpression was performed by transfecting the miRNA expression plasmid clearly using transfection reagent. The empty vector was used as vehicle control. miR 32 overexpression was confirmed by qPCR using TaqMan probes specific to miR 32. Reverse transcription and real time PCR RNA was subjected to DNAse treatment for 30 minutes at 37 C. cDNA synthesis was performed with reverse tran scriptase in accord ance with the manufacturers protocol.
PCR conditions were, 65 C for 5 minutes, 25 C for 10 minute, 42 C for 50 minutes, and 70 C for 10 minutes, and finally RNAse H treatment for 20 minutes at 37 C. SYBR Green was used for qPCR. The primer sequences used in the study for qPCR are listed in Table 1. Transfection with anti microRNA CHME3 cells were Inhibitors,Modulators,Libraries transfected with 100 pmol of anti Inhibitors,Modulators,Libraries miR 32 and Cy3 labeled control anti miR. After 48 hours of transfec tion, cells were pelleted for RNA isolation and protein lys ate preparation. Transfection efficiency was assessed by visualizing the fluorescence of Cy3 labeled control anti miR. Knockdown of miR 32 in anti miR transfected cells was assessed using an miR 32 assay. TRAF3 protein ex pression in cells transfected with anti miR 32 was ana lyzed using western blotting with anti TRAF3 antibody.
Luciferase reporter assay Inhibitors,Modulators,Libraries HeLa cells were seeded in six well plates and co transfected with luciferase reporter clones of TRAF3 3 UTR and a miR 32 expressing plasmid. The TRAF3 3 UTR con struct in pMirTarget and miR 32 construct as pCMV Mir were used. A mutation in the 3 UTR of TRAF3 in miR 32 binding sites was created by deleting the TATT sequence at position 463 to 466 of the 3 UTR using the primer set listed in Table 1. A site directed mutagenesis kit was used for generating the deletion mutations. Both the wild type and mutant 3 UTR of TRAF3 were transfected along with miR 32 ex pression clones in HeLa cells. Cells were harvested after 24 hours of transfection for luciferase assays in accordance with the manufacturers proto Inhibitors,Modulators,Libraries col. A B galactosidase assay was used for normalization.
Statistical analysis Results are shown as the mean and standard error of the mean from Inhibitors,Modulators,Libraries three independent repeated experiments. Results are shown relative to controls in miR 32 assay. The level of significance between treated and untreated groups was analyzed using the Stu dents t test, and P 0. 05 was considered significant. Results Expression and purification of tat protein We could express the recombinant HIV 1 Tat C protein successfully by using the standard procedures selleckchem Axitinib as described in material and methods section.