Figure 6 | Oncogene

Figure 6

From: Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE

Figure 6

c-Src phosphorylates Lpd and the Lpd-Scar/WAVE interaction is positively regulated by c-Abl and c-Src. (a) HEK293FT cells were transfected with either GFP as a control or GFP-Lpd and co-transfected with Src-WT (wild type) or Src-KI (kinase inactive). Immunoprecipitation was performed from cell lysates using Lpd-specific antibodies or rabbit IgG as a control followed by western blotting with anti-Lpd and anti-phosphotyrosine (pTyr) antibodies. n=3. (b) Abl and Arg double knockout MEFs (Abl/Arg DKO) were serum starved overnight and treated with 10 μm Bosutinib (c-Src kinase inhibitor) for 2 h before stimulating with 20 ng/ml PDGF-BB for 2 min. Immunoprecipitation was performed from cell lysates using Lpd-specific antibodies or rabbit IgG as a control followed by western blotting with anti-Lpd and anti-phosphotyrosine (pTyr) antibodies. (c) Quantified band intensities of chemiluminescence blots from (b) of Lpd and pTyr imaged with a CCD camera. pTyr was normalized against the immunoprecipitated Lpd. The pTyr signal from Rabbit IgG control lanes was subtracted from the pTyr signal from the immunoprecipitated Lpd lanes. n=3, data are represented as mean±s.e.m. One-way ANOVA; Dunnett's; ***P0.001, ****P0.0001, NS, not significant. (d) HEK293FT cells were transfected with GFP-Lpd, Myc-tagged components of the Scar/WAVE complex and either Src-WT (wild type) or Src-KI (kinase inactive). Immunoprecipitation was performed from cell lysates using Lpd-specific antibody or rabbit IgG as a control followed by western blotting with anti-GFP, anti-Myc and anti-phosphotyrosine (pTyr) antibodies. (e) Quantified band intensities of chemiluminescence blots (d) of GFP-Lpd and Myc-tagged components of the Scar/WAVE complex imaged with a CCD camera. Individual Scar/WAVE components were normalized against the immunoprecipitated Lpd. n=4, data are represented as mean±s.e.m. One-way ANOVA; Dunnett's; *P0.05, **P0.01. (f) HEK293FT cells were transfected with GFP-Lpd, Myc-tagged components of the Scar/WAVE complex and either GFP-c-Abl or GFP. Immunoprecipitation was performed from cell lysates using Lpd-specific antibody or rabbit IgG as a control followed by western blotting with anti-GFP, anti-Myc and anti-phosphotyrosine (pTyr) antibodies. (g) Quantified band intensities of chemiluminescence blots (f) of GFP-Lpd and Myc-tagged components of the Scar/WAVE complex imaged with a CCD camera. Individual Scar/WAVE components were normalized against the immunoprecipitated Lpd. n=4, data are represented as mean±s.e.m. One-way ANOVA; Dunnett's; **P0.01, ***P0.001.

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