Figure 2. Capz increases protein tyrosine phosphatase epsilon (PTPε) levels in prostate cancer cells.

A. DU145 cells (1 × 10⁶ cells/well) were treated with the indicated concentrations of pervanadate and 5 μM Capz for 6 h. Whole-cell extracts were prepared and immunoblotted with antibody for p-STAT3(Tyr705). The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. The results shown are representative of two independent experiments. B. DU145 cells (1 × 10⁶ cells/well) were treated with 5 μM Capz for the indicated durations (left) or treated for 6 h and washed with PBS twice to remove Capz before resuspension in fresh medium (right). Cells were removed at indicated times and lysed to prepare whole-cell extracts. C. DU145 cells (1 × 10⁶ cells/well) were treated with the indicated concentrations of Capz for 6 h. Whole-cell extracts were prepared and immunoblotted with antibody for PTPε. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown are representative of two independent experiments. D. DU145 cells (1 × 10⁶ cells/well) were treated with the indicated concentrations of Capz for 6 h. Total RNA was extracted and PTPε C and PTPε M expression were examined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to verify equal RNA loading. E. DU145 cells (1 × 10⁶ cells/well) were transfected with either scrambled or PTPε-specific siRNA (50 nM). After 48 h, cells were treated with 5 μM Capz for 6 h. Whole-cell extracts were prepared and immunoblotted with antibody for PTPε. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown are representative of three independent experiments. F. Equal amounts of lysates were analyzed by Western blot using antibody against p-STAT3(Tyr705). The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. G and H. DU145 cells (1 × 10⁶ cells/well) were transfected with either scrambled or PTPε-specific siRNA (50 nM). After 48 h, cells were treated with 5 μM Capz for 24 h. Whole-cell extracts were prepared and immunoblotted with antibodies for MMP-9 and PARP. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. I. LNCaP cells (1 × 10⁶ cells/well) were treated with 5 μM Capz for 6 h and then stimulated with IL-6 (25 ng/mL) for the indicated times. Whole-cell extracts were prepared and immunoblotted with antibodies for p-STAT3(Tyr705) and STAT3. J. Equal amounts of lysates were analyzed by Western blot using antibodies against p-Src(Tyr416) and Src.