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. 2009 Nov 17;122(24):4439–4451. doi: 10.1242/jcs.051847

Fig. 2.

Fig. 2.

Knockdown of Src attenuates whereas overexpression of Src enhances Wnt/β-catenin signaling. (A) siRNA targeting Src inhibits Wnt3a activation of Lef/Tcf-sensitive transcription. F9 cells were treated with siRNAs targeting Src for 1 day before co-transfection of the cells with rat Fz1 and Super8xpTOPFlash plasmids. Cells were stimulated with or without Wnt 3a for 8 hours. The activity of the luciferase reported gene was assayed and the transcriptional activity displayed relative to the unstimulated cells (set to `1'). The results show mean values ± s.e.m. obtained from five separate experiments. Cell lysates were analyzed by immunoblotting; blots were stained with anti-Src antibody (or anti-GAPDH antibody as a loading control). (B) siRNA targeting Src inhibits Wn3a activation of PE formation. F9 cells were transfected with siRNA targeting Src 1 day before rat Fz1 transfection. Cells were treated with or without Wnt3a for 3 days and cultured for a further 2 days. Blots that were stained with the PE marker TROMA-1 antibody were quantified by the calibrated scanner and results were displayed as `fold' (time = 0, set to `1'). The results shown are mean values ± s.e.m. from ten independent experiments. (C) Overexpression of Src enhances Wnt3a-sensitive Lef/Tcf-sensitive transcription. F9 cells were co-transfected with rat Fz1, Super8xTOPFlash reporter, and a Src expression vector one day before cells were stimulated without or with purified Wnt3a for 8 hours. Cell lysates were assayed for Lef/Tcf luciferase transcription activity. Results are displayed relative to the unstimulated cell (set to `1'). The results are shown as mean values ± s.e.m. from six independent experiments. Statistical significance is indicated (*P<0.05; **P<0.01). Right panels display cellular content of Src and of GAPDH (as loading control). Representative immunoblots are shown. (D) Overexpression of Src alone stimulates β-catenin accumulation. Control (–) and Src-overexpressing (Src) F9 cells were lysed with RIPA buffer and cell lysates were analyzed for β-catenin content by immunoblotting with anti-β-catenin antibody or anti-GAPDH antibody (loading control). Blots representative of three separate experiments are displayed.