tPA inhibits LPS-initiated cell-signaling in macrophages. (A) BMDMs from WT C57BL/6J mice were treated with LPS (0.1 µg/mL) alone for 1 h or simultaneously with h-EI-tPA (tPA, 12 nM), activated α2M (10 nM), or MMP9-PEX (PEX, 10 nM). (B) BMDMs were treated with LPS (0.1 µg/mL) and with increasing concentrations (0.2-24 nM) of h-EI-tPA for 1 h. (C) BMDMs were treated with LPS (0.1 µg/mL) and with increasing concentrations (0.2-24 nM) of h-EA-tPA for 1 h. (D) BMDMs were treated with LPS (0.1 µg/mL) and with increasing concentrations (0.2-24 nM) of m-EI-tPA for 1 h or with 12 nM h-EI-tPA as a control. Equal amounts of cellular protein (20 µg) were subjected to immunoblot analysis to detect phospho-IκBα, total IκBα, phospho-ERK1/2, and total ERK1/2. (E) BMDMs were treated for the indicated times with LPS (0.1 µg/mL). Cell extracts were subjected to immunoblot analysis to detect phospho-IκBα, total IκBα, and β-actin. (F) Densitometry analysis of 4 independent experiments in which we examined IκBα phosphorylation in BMDMs treated with LPS for the indicated times (mean ± SEM). The analysis was performed using Image Studio Lite 5.2. (G) BMDMs were treated for the indicated times with LPS (0.1 µg/mL) and h-EI-tPA (12 nM). Cell extracts were subjected to immunoblot analysis to detect phospho-IκBα, total IκBα, and β-actin. p-ERK, phospho-ERK1/2; t-ERK, total ERK1/2; Veh, vehicle.
