Figure 5. ETEC activation of NF-κB depends upon Rap1 activation.

A. Immunoblotting of phosphorylated IκBα after treating HCT-8 cells with TNF-α (20 ng/ml, 30′) or infecting them with either wt or ΔeltA ETEC for 2 h. Where indicated, HCT-8 cells were first treated with DDA (200 μM, 1 h). B. Quantification of data shown in panel A. Data are presented as mean ± SEM, n = 3, with asterisks (*) used to indicate a statistically significant difference from wt ETEC (p < 0.05, Student’s t test). C. Immunoblotting of GTP-bound (activated) and total Rap1 after treating HCT-8 cells with forskolin (10 μM, 1 h) or infecting them with either wt or ΔeltA ETEC for 2 h. D. Immunoblotting of phosphorylated IκBα after transfecting cells with a Rap1-GAP1 expression plasmid. The numbers above the immunoblots indicate normalized phosphorylated IκBα abundance. E. Immunoblotting of phosphorylated IκBα, nuclear p65, and Rap1-GAP1 after infecting HCT-8 cells with either wt or ΔeltA ETEC for 2 h, in the presence or absence of Rap1-GAP1 or empty vector. F. Quantification of IκBα phosphorylation and nuclear p65 levels shown in panel E. Data are presented as mean ± SEM, n = 3–4, with asterisks (*) used to indicate a statistically significant difference from wt ETEC (p < 0.05, Student’s t test). G. Immunoblotting of total and phosphorylated ERK1/2, p38, and JNK after infecting HCT-8 cells with wt ETEC for 2 h, in the presence or absence of Rap1-GAP1 or empty vector. Phosphorylated protein abundance was normalized to total protein abundance. H. Quantification of data shown in panel G. I. Immunoblotting of total and phosphorylated ERK1/2, p38, and JNK, phosphorylated IκBα, Rap1, and tubulin after treating HCT-8 cells with TNF-α (20 ng/ml, 30′) or infecting them with either wt or ΔeltA for 2 h. Where indicated, HCT-8 cells were first transfected (48 h prior to infection) with an siRNA duplex targeting Rap1 or a non-specific (ns) siRNA duplex.