Figure 11.

Analysis of expression of Egr2, IL-6, and TNF on a protein level in activated macrophage cell line with overexpression of Egr2. Macrophage cell line RAW264.7 was transfected with empty (control) pMIG plasmid vector (Control Vector), or vector with Egr2 (Egr2 Vector) for 24 h and after which the cells were activated with IL-4 or IFNγ for another 24 h-time period as in Figure 10. The cells were washed, fixed/permeabilized and stained for intracellular Egr2, IL-6, or TNF with fluorescently labeled mAbs as described in Materials and Methods. GFP⁺ gated transfected cells were analyzed for expression of Egr2, IL-6, or TNF by three-color flow cytometry. Expression of Egr2 (A,B), IL-6 (C,D) and TNF (E,F) in cells transfected with Egr2 Vector (solid line) vs. Control Vector (dotted line) are shown on representative histogram graphs (A,C,D). Staining with isotype-matched control mAbs is shown by shaded histograms. Quantitative analysis (mean fluorescence intensity level for Egr2 and percentage of positive cells for IL-6 and TNF) is shown in (B,D,F). In (B,D,E), mean ± S.E. of six separate culture plate wells is shown. Statistically significant differences in the expression levels are shown on each figure (^*p < 0.05; ^***p < 0.001; ^****p < 0.0001).