FIGURE 3.

N-Ras activation is dependent on TLR4 but not TRAF6. A, H69 cells were transfected with TLR4-siRNA (50 nm), scrambled (Scr)-siRNA (50 nm), or FuGENE HD alone (control, Ctrl) 24 h prior to treatment. The cells were treated with LPS (200 ng/ml) for 15 min, and the RAS activation assay was performed. Ponceau Red stain was utilized to detect RBD-GST to confirm equal loading. N-Ras activation was observed following LPS treatment; however, the TLR4-siRNA diminished N-Ras activation compared with both LPS-treated control and LPS-treated cell transfected with the scrambled control (Scr siRNA). B, following a 15-min LPS treatment, an increase in phosphorylated ERK is detected in H69 cells. However, transfection of H69 cell with an N-Ras siRNA, which effectively depleted N-Ras, blocked LPS-induced ERK phosphorylation. C, stable transfections of H69 cells, using three different TRAF-6 shRNA constructs (#77, 78, and 80) or with the pGIPZ empty vector were performed. Following stable clone selection, immunoblotting was performed for TRAF6. The 60-kDa TRAF6 protein was detected in the pGIPZ-transfected cells, but TRAF6 was largely diminished in the shRNA expressing cell lines. D, the NFκB luciferase reporter system was utilized to confirm the functional depletion of TRAF6 in the cells expressing shRNA #80. Cholangiocytes stably transfected with either the pGIPZ empty vector (black bars) or TRAF6-shRNA (gray bars) were treated with LPS (200 ng/ml) or HKLM (1 × 10⁸ cells/ml) for 5 h followed by the Dual Luciferase Reporter assay. Both LPS and HKLM induced NFκB-driven luciferase in pGIPZ-transfected cells (*, p < 0.05 compared with control untreated cells), whereas the TRAF6-shRNA-expressing cells showed no increase in NFκB-driven luciferase activity. Data are presented as mean ± S.E. (error bars). E, an N-Ras activation assay was also performed on LPS-treated TRAF6-depleted cells. The pGIPz control transfected or TRAF6-depleted cells were treated with LPS or HKLM for 15 min. N-Ras activation was not diminished in those cells depleted of TRAF6. The membrane was stained with Ponceau Red to confirm equal loading. EV, empty vector. F, depletion of TRAF6 had no effect on LPS-induced ERK phosphorylation. LPS (200 ng/ml for 15 min) treatment of both the pGIPZ empty vector control cells and TRAF6-depleted cells resulted in a similar increase in phosphorylation of ERK1/2 as demonstrated by Western blotting.