Figure 6. TCPTP regulates TβRII tyrosine phosphorylation and signaling.

(A) Cell lysates (0.5 mg) from serum-starved WT and α1KO CD cells were immunoprecipitated with the anti-phosphotyrosine antibody 4G10 (10 μg) or with mouse IgG isotype control antibody (10 μg) and analyzed by Western blot. A band corresponding to TβRII (~68 kDa) was visible only in α1KO cells incubated with 4G10. Tyrosine-phosphorylated products (50–100 kDa) were detected with anti-pY99 antibodies in both WT and α1KO CD cells. (B) Morphology of WT CD cells stably transfected with shRNAi control (ShC) or TCPTP shRNAi (Sh-TCPTP). (C and D) Cell lysates (20 μg/lane) from serum-starved WT CD cells transfected with ShC (1 clone shown) or Sh-TCPTP (3 clones shown) and treated with or without SB431542 (SB) were analyzed by Western blot for levels of TCPTP, pSMAD3, SMAD3, and collagen I. (E) WT CD cells were cultured on plastic in 0.2% serum with or without the TCPTP inhibitor compound 8 (TCPTP-I) for 4 days and then stained with anti–ZO-1 and anti-αSMA antibodies. (F) Serum-starved WT CD cells were treated with TCPTP inhibitor at the concentrations indicated. After 24 hours, cell lysates (20 μg/lane) were analyzed for levels of collagen IV, pSMAD3, and SMAD3. (G) Cell lysates (0.5 mg) from serum-starved WT CD cells transfected with control or TCPTP shRNAi (1 clone each shown) were immunoprecipitated and analyzed by Western blot as in A. A band corresponding to TβRII was more evident in lysates of CD cells transfected with TCPTP shRNAi. Scale bars: 20 μm (B and E).