tPA independently activates cell signaling in macrophages. (A) BMDMs from WT C57BL/6J mice were treated with h-EI-tPA (12 nM), in the absence of LPS, for the indicated times. Immunoblot analysis was performed to detect phospho-IκBα, total IκBα, and β-actin. (B-E) BMDMs were treated with LPS (0.1 μg/mL), h-El-tPA (12 nM), activated α₂M (10 nM), LPS plus h-EI-tPA, LPS plus activated α₂M, or vehicle for 8 h. RT-qPCR was performed to compare mRNA levels for TNFα, IL-1β, IL-6 and CCL3 (mean ± SEM; n = 8; 1-way ANOVA with Tukey’s post hoc analysis). (F) BMDMs were treated for 4 or 8 h with LPS alone (0.1 μg/mL), h-EI-tPA alone (12 nM), or LPS plus h-EI-tPA. Cell extracts were subjected to immunoblot analysis to detect phospho-IκBα, total IκBα, and β-actin. *P < .05, ***P < .001.