Figure 2

SHP-1 has an important role in pectolinarigenin-mediated STAT3 Tyr705 phosphrylation repression. (a) 143B and MG63.2 cells were treated with vanadate (100 μM) and pectolinarigenin (20 μM). Whole-cell lysates were prepared and applied to immunoblotting with an anti-phospho-STAT3 (Y705) antibody. (b) 143B cells were exposed to increasing concentrations of pectolinarigenin. SHP-1, SHP-2 and PTEN were probed by a western blot assay. (c) 143B cells were treated with pectolinarigenin for 24 h and then analyzed for SHP-1 mRNA expression by RT-PCR. (d) 143B cells were treated with pectolinarigenin and nuclear extracts were prepared. The immunoprecipitation assay was performed using indicated antibodies. (e) 143B cells were incubated with pectolinarigenin and the proteins were cross-linked with DNA and analyzed by a quantitative ChIP assay with indicated antibodies. (f) 143B cells were transfected with SHP-1 siRNA and treated with pectolinarigenin (20 μM). Western blot analysis was used for detecting phospho-Tyr705-STAT3 expression. Cell viability was measured by a MTS assay (**P<0.01). (g) SHP-1 was overexpressed in 143B cells. Cells were then incubated with 20 μM pectolinarigenin. Phospho-Tyr705-STAT3, STAT3 and SHP-1 were detected by a western blot assay. Cell viability was measured by a MTS assay (***P<0.001)