FIGURE 4.

Transcriptional regulation of SOCS proteins. Macrophages were infected with L. donovani for the indicated time periods. One group of infected macrophages from each time point was subjected to H₂O₂ treatment for 1 h. A and B, Egr1 expression was determined at the mRNA level (A) and protein level (B) by real time PCR and Western blotting respectively. C, cells were stained with anti-Egr1 monoclonal antibody followed by secondary Texas Red-conjugated antibody. Nuclei were stained with DAPI, and cells were analyzed under fluorescence microscope. D, E, G, and H, labeled Egr1 probe (D and G) or Egr1 probe with a mutated binding site (E and H) was incubated with nuclear extracts prepared from cells treated as above. DNA binding was analyzed by EMSA. F, cells were treated as above; nuclear and cytosolic extracts were prepared, and expression of Egr1 was analyzed by immunoblotting. I and J, cells were infected with L. donovani for the indicated time periods and analyzed for Egr1 ChIP assay. PCR amplification of anti-Egr1 immunoprecipitates (IP) and total input chromatin are shown in the upper and lower panels, respectively. K and L, macrophages were transfected (24 h) with either control or Egr1 siRNA followed by infection with L. donovani promastigotes for 6 h. Expression of Egr1 (K) and SOCS1 and SOCS3 (L) was evaluated by immunoblot analysis. Results are representative of three individual experiments, and the error bars represent mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student's t test.