Figure 6.

Preemptive SOS p65 siRNA treatment of mice inhibited glomerular NF-κB activation by EMSA and NF-κB–dependent TNF-α mRNA expression by quantitative RT-PCR that correlated with crescent formation. (A) Glomeruli were prepared from two control-treated (SOS ctrl) and two SOS p65 siRNA-treated (SOS siRNA) mice and NF-κB was assessed by EMSA. (B) Nuclear kidney extracts from SOS ctrl (black, n=5) and SOS siRNA (gray, n=5) mice were subjected to EMSA and the OD of the p50/65 heterodimer was assessed. (C) mRNA was prepared from the kidney of each mouse shown in (B), TNF-α mRNA expression was determined by qRT-PCR and plotted against the percentage of crescents. (D) Costaining using fluorescence-labeled antibodies to the EC marker CD31 (red), phospho-p65 (green), and DAPI staining for nuclei (blue) was performed in renal tissue from SOS ctrl– and SOS siRNA–treated mice. A typical microscopy example (original magnification, ×40) together with the corresponding quantitative assessment of nuclear phospho-p65 staining within CD31-positive glomerular ECs using Image J is depicted. **P<0.01.