Notes: HEK 293T cells were transfected with a reporter plasmid (assembled in the pGL3 Basic Vector) with a consensus BCL6 binding site and a BCL6 expression plasmid using the ViaFect™ Transfection Reagent (about 2µg of DNA per well in 6-well plates at 50-60% confluency). Promoter activation was monitored with a luciferase assay control with a β-galactosidase control. Western blotting of transfected cells used the Anti-Rabbit IgG (Fc), Alkaline Phosphatase Conjugate and the Western Blue® Stabilized Substrate for Alkaline Phosphatase. (4657)

Notes: To test the feasibility of the Mycobacterium bovis Ag85B gene as a DNA vaccine, the gene was amplified by PCR, introducing EcoRI and XbaI restriction endonucleases onto the primers, digested with the restriction enzymes and ligated into the pCI Mammalian Expression Vector. After transformation, the constructs were isolated using the Wizard^® Plus Minipreps DNA Purification System and analyzed using endonuclease digestion and DNA sequencing. To confirm production of a maltose binding protein (MBP)-Ag85B fusion protein in E. coli, the cell lysate was analyzed by Western blot, stained with a rabbit anti-MBP serum and secondary antibody Anti-Rabbit IgG (Fc), AP Conjugate (1:10,000 dilution) and developed with BCIP/NBT. BALB/c mice pretreated with 10mM cardiotoxin prior to intramuscular DNA immunization using construct pCI-Ag85B, administered at days 0, 15, 30, and 45. The immunization course was 50µl of DNA at a concentration of 1µg/µl in PBS with each animal receiving a total of 100µg of plasmid DNA. Antibody response was assessed at 15 days with an ELISA assay and cytokine presence tested two weeks after immunization using splenocytes for ELISA assays and ICC. (3552)

Notes: The ability of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to upregulate cytochrome P450 CYP1A1 levels in mouse liver was examined. Total mouse liver RNA was reverse transcribed using the Reverse Transcription System, and the resulting CYP1A1 cDNA was quantitated using real-time PCR. CYP1A1 protein levels were quantitated by Western blot using an anti-CYP1A1 antibody, the Anti-Rabbit IgG (Fc), Alkaline Phosphatase Conjugate secondary antibody and the Western Blue^® Stabilized Substrate for Alkaline Phosphatase.
(3443)

Notes: Immunoblots, on PVDF, were developed with the Anti-Rabbit IgG Alkaline Phosphatase Conjugate and the Western Blue^® Stabilized Substrate for Alkaline Phosphatase. (1200)

Notes: Poly(A)+ RNA was purified with PolyATtract^® mRNA Isolation Systems. Anti-Rabbit IgG AP Conjugate, and BCIP/NBT were used in Western blotting. R transcripts were synthesized from the Lc cDNA in the pGEM-7Z(+) Vector, and were used in in vitro translation by Rabbit Reticulocyte Lysate Systems to be used as a positive control in Western. (0781)