FIG 6.

SH2B1β regulates Cdh2 promoter activity through STAT3 and Sp1. (A) PC12-GFP and PC12-SH2B1β cells were treated with 100 ng/ml FGF1 for the indicated number of days. Cell lysates were collected, and equal amounts of proteins were separated by SDS-PAGE and immunoblotted with anti-N-cadherin, anti-GAPDH, and anti-α-tubulin antibodies. GAPDH or α-tubulin was used as a loading control. The level of N-cadherin was normalized to GADPH or α-tubulin. Values of PC12-GFP cells are the means ± standard deviations from two independent experiments, and values of PC12-SH2B1β cells are the means ± SEM from three independent experiments. (B) PC12-SH2B1β cells were preincubated with DMSO or STA-21 (20 μM) for 1 h and then were left untreated or treated with 100 ng/ml FGF1 for 4 days. Cell lysates were analyzed by Western blotting using anti-N-cadherin and anti-GAP-43 antibodies. α-Tubulin was used as a loading control. The level of N-cadherin was normalized to α-tubulin. Values are the means ± SEM from three independent experiments. (C) PC-3 cells were transiently transfected with SH2B1β, STAT3, or Sp1 together with Cdh2 promoter sequences fused to firefly luciferase and pEGFP. Cells were harvested 18 h later, and luciferase activities were measured. Firefly luciferase activities were normalized to pEGFP levels. (D) PC-3 cells were transiently cotransfected with SH2B1β ± STAT3 (left), SH2B1β ± Sp1 (middle), or STAT3 ± Sp1 (right), together with the Cdh2 promoter construct and pEGFP. Cells were harvested at 18 h, and luciferase activities were analyzed as described for panel C. (E) PC-3 cells were transiently transfected with Sp1 along with vector control, STAT3, and STAT3-C-FLAG (designated WT and C). Cell lysates were extracted and immunoprecipitated using anti-Sp1 antibody and analyzed by Western blotting using antibodies against Sp1, STAT3, and N-cadherin. (F) PC-3 cells were transiently transfected with GFP or GFP-SH2B1β plasmid. Cell lysates were immunoprecipitated using anti-GFP antibody and analyzed by Western blotting using antibodies against SH2B1 and Sp1. (G) PC-3 cells were left untransfected or were transiently transfected with STAT3 plus Cdh2 reporter constructs and pEGFP. Cells were treated with 0, 1, or 5 μM MMA for 18 h and then were harvested for luciferase activity measurements. Firefly luciferase activities were normalized to pEGFP levels. Values in panels C, D, and G are means ± SEM from three independent experiments. *, P < 0.05 by paired Student's t test.