FIG. 2.

Concentration (PFU) and time dependence of in vitro NF-κB activation in endothelial cell cytoplasmic extracts. (A) Aliquots of cytoplasmic extracts from unstimulated endothelial cells (cyt) were incubated at 37°C for 30 min with undiluted R. rickettsii (RR) along with R. rickettsii diluted 1:5, 1:20, and 1:100 (RR/5, RR/20, and RR/100, respectively), followed by EMSA. Lanes marked +cold indicate the presence of a 10-fold molar excess of unlabeled probe in the binding reaction mixture. Cytoplasmic extract was also treated with NP-40 and DOC as described for Fig. 1A. NS, nonspecific complex. (B) Endothelial cell cytoplasmic extracts were incubated with approximately 10⁵ PFU of partially purified R. rickettsii for 15, 30, 60, and 120 min, followed by EMSA. The autoradiogram of the gel was scanned as described in Materials and Methods, and band intensities were corrected by subtraction of baseline levels of NF-κB in cytoplasmic extracts and R. rickettsii preparations alone.