Figure 6

Cell fractionation analysis shows significant increased level of phospho-STAT3 in the nucleus (N) but not cytoplasm (C) on LPS-treated DCs with Shc silencing. DCs from Lewis rats were stimulated with LPS (0.5 μg/ml) in the presence of Shc siRNA (50 nM) or equal amount of NEG. siRNA. Nuclei were pelleted and the supernatant was collected as described in Materials and Methods. The cytoplasmic fraction (C) and the nuclear fraction (N) were analyzed by SDS/PAGE and Western blot for the presence of endogenous STAT3. The phosphorylation levels of endogenous STAT3 at Tyr-705 were significantly increased in both N and C fractions after LPS treatment for 6 h. However, the significant enhancement of STAT3 phosphorylation was observed only in the N fraction LPS-treated DCs in the presence of Shc siRNA. Each column represents the mean ± SE of the three independent experiments (n = 3; *, P < 0.05).