Figure 2.
HMGB1 induces NF-κB activation via an ERK- dependent mechanism. (A) 3T3 fibroblasts were stimulated with 100 ng/ml HMGB1 or 10 ng/ml PDGFBB for 5, 15, 30, or 60 min, and total cell proteins were analyzed by Western blot. ERK1/2 activation was visualized with anti–phospho- ERK antibody (pERK1/2). Stripped blots were reprobed with antibodies against total ERK (ERK1/2). Results are representative of three independent experiments. (B) U0126 inhibits HMGB1-induced cell migration. 3T3 fibroblasts were pretreated with or without 10 μM of the MEK1/2 inhibitor U0126 for 30 min and were allowed to migrate in Boyden chambers for 3 h toward 30 ng/ml HMGB1 or no stimulus. Each error bar represents the mean ± SD of triplicate samples. This experiment was repeated three times. (C) HMGB1 induces NF-κB nuclear translocation. 3T3 fibroblasts grown on coverslips were serum starved for 16 h and stimulated with 100 ng/ml HMGB1 or 20 ng/ml TNF-α for 20 and 10 min, respectively. Cells were fixed and immunostained indirectly for p65/RelA. (D) IκBα is phosphorylated in 3T3 fibroblasts exposed to HMGB1 or TNF-α. Cells were lysed in SDS-PAGE loading buffer at the indicated times; proteins were Western blotted with rabbit polyclonal antibodies against mouse pIκBα or mAb against β-actin. (E) MEK inhibitor U0126 prevents NF-κB nuclear translocation. 3T3 fibroblasts grown on coverslips were serum starved for 16 h, exposed where indicated to U0126, and stimulated 30 min later with 100 ng/ml HMGB1. After 20 min, cells were fixed and immunostained indirectly for p65/RelA. (F) The MEK inhibitor U0126 impairs IκBα phosphorylation. 3T3 fibroblasts were exposed to 30 μM U0126 for 30 min and stimulated with 100 ng/ml HMGB1 for 20 min. Total cellular proteins were subjected to Western blotting with rabbit polyclonal antibodies against mouse pIκBα or mAb against β-actin.