Figure 4

Inhibitory effect of CTS on p-STAT3 is mediated by SHP-2. (a) U251 cells were treated with pervanadate at indicated concentration for 1h, and with additional 10 μM CTS for 2 h. Proteins were analyzed by western blotting with STAT3, p-STAT3 (Tyr705) and GAPDH antibodies. (PI: phosphoesterase inhibitors, pervanadate). (b) U251 cells were treated with 10 μM CTS for 0, 0.15, 0.5, 1, 2, 4 h. Proteins were analyzed by western blotting with SHP-1, TC-PTP, SHP-2, p-SHP-2 (Tyr542), p-SHP-2 (Tyr580) and GAPDH antibodies. (c–e) U251 cells were transfected with specific phosphates siRNA including SHP-1 (c), TC-PTP (d), SHP-2 (e) or negative control (NC) for 48 h, and then treated with CTS with the indicated concentration for 120 min, followed by western blot analysis with the indicated phosphates antibodies. Expression of p-STAT3 (Tyr705) relative to STAT3 were quantified by densitometry. Results are the mean±S.E.M. (n=3 for each group). ^##P<0.01, ^###P<0.001 versus control. (f) Cell lysates from U251 cells treated with 0, 5, 10 μM CTS for 2 h were subjected to SHP-2 phosphatase activity assay. Data are expressed as mean±S.E.M., n=3 for each group. *P<0.05; **P<0.01 versus CTS 0 μM group. (g and h) U251 cells were pretreated with 25 μM NSC-87877 for 30 min, followed by 10 μM CTS treatment for 120 min. Total cell lysates were prepared and subjected to western blot analysis with indicated antibodies (g) or SHP-2 phosphatase activity assay (h). Data are expressed as mean±S.E.M., n=3 for each group. *P<0.05; **P<0.01, versus CTS 0 μM group