Figure 2
Ligand-independent interaction of Necl-4 with ErbB3. (a) Ligand-independent interaction of Necl-4 with ErbB3. HEK293E cells were co-transfected with various combinations of the indicated plasmids and cultured in the presence or absence of GST-HRG. FLAG-Necl-4 was immunoprecipitated using the anti-FLAG mAb, and the samples were subjected to Western blotting using the indicated Abs. Arrowhead, co-immunoprecipitated ErbB3. Square bracket, immunoprecipitated FLAG-Necl-4. (b) In vitro GST-HRG-binding assay. HEK293E cells were co-transfected with FLAG-Necl-4 and ErbB3-GFP or FLAG-Necl-4 alone as indicated at the bottom. The Necl-4-ErbB3 complex was immobilized on the beads using the anti-FLAG mAb and further incubated with 0.3 μM recombinant GST or GST-HRG at 25 °C for 30 min. The precipitated proteins with the beads were subjected to Western blotting using the indicated Abs. Arrowhead, co-precipitated GST-HRG; square bracket, co-immunoprecipitated Necl-4; double-arrowhead, immunoprecipitated ErbB3. (c) No effect of Necl-4 on the GST-HRG binding to ErbB3. HEK293E cells were co-transfected with ErbB3-GFP and different amounts of FLAG-Necl-4 as indicated at the bottom, and the cells were cultured in the presence or absence of 30 nM GST-HRG at 37 °C for 10 min. ErbB3-GFP was immunoprecipitated using the anti-GFP pAb, and the precipitated proteins with the beads were subjected to Western blotting using the indicated Abs. Arrowhead, co-precipitated GST-HRG; square bracket, co-immunoprecipitated FLAG-Necl-4; double-arrowhead, immunoprecipitated ErbB3-GFP. IB, immunoblotting; IP, immunoprecipitation.
