Figure 3.

In vivo evaluation of genetically engineered SOFA-HDV Rz to suppress the expression levels of the human Pax-5B gene products. (A) Northern analysis were conducted on RNA extracts from 293T cells transiently cotransfected with Pax-5B (500 ng); increasing amounts of SOFA-HDV Rz (1, 100 and 1000 ng) and finally filler DNA to totalize 1.5 μg. Individual controls are indicated as the untransfected cells (293T); cells cotransfected with Pax-5B along with the SOFA-HDV Rz empty vector (identified Pax-5B); and cells cotransfected with SOFA-HDV Rz along with the Pax-5B empty vector (identified Rz). Radiolabeled RNA probes were then hybridized onto the membrane to reveal transcript expression levels from intracellular Pax-5B (top panel) and actin (bottom panel) as an internal reference. (B) The radioactivity from the hybridized Pax-5B probes was then calculated and normalized on actin transcripts densities using a radioanalytic scanner and plotted with error bars in relation to expression ratios (in black). SOFA-HDV Rz RNA levels are also plotted accordingly (right of the panel, in white). (C) Electrophorectic Mobility Shift Assays (EMSA) were conducted on nuclear extracts from 293T cells cotransfected with Pax-5B and SOFA-HDV Rz. Nuclear extracts were incubated with either with Pax-5 (top panel) or NF-κB-specific (bottom panel) dsDNA oligonucleotides labeled at the 5′ end with [γ-³²P]. DNA–protein complexes were resolved and analyzed on a 4% polyacrylamide gel. Specific (S) and non-specific (NS) cold competition (Comp) assays were carried out by adding a 100-fold molar excess of unlabeled dsDNA oligonucleotides corresponding to the Pax-5 or the NF-κB consensus-binding motif. Control lanes were loaded with free probe (Probe) and extracts from untouched 293T cells (293T). Other controls include transfections of the Pax-5B empty vector (identified pcDNA), the Pax-5B expression vector (identified Pax-5B) and finally the SOFA-HDV Rz empty vector (identified psiRNA).