Skip to main content
. 2012 Nov 23;20(3):443–455. doi: 10.1038/cdd.2012.136

Figure 1.

Figure 1

CCN2 binds to EGFR though the carboxyl-terminal CT domain. (a) Western blot analysis of CCN2 in CL1-5, A549, CL1-0 cells transduced with either CCN2 or siCCN2 and the corresponding control vectors as indicated. α-tubulin levels were used as loading control. One representative experiment of three is shown. (b) CCN2 bound to EGFR was FACS analyzed in control siRNA, or siRNA for EGFR depletion (siEGFR) transfected CL1-5 (left) or A549 (right) cells. (c) A549 cells were cotransfected with CCN2- and EGFR-expression plasmids (top left) or transfected with EGFR-expression plasmid and treated with rCCN2 (100 ng/ml) (bottom left), and then incubated with rEGF (20 ng/ml) for 24 h. Cells were collected and western-immunoprecipitation analysis (WB-IP) was performed to detect CCN2 and EGFR expression as indicated. In vitro recombinant protein: CCN2 and EGFR were mixed and subjected to western-immunoprecipitation analysis (right). Immunoblotting showed the resulting expression and monitored for expression of EGFR and CCN2 complex. The data was represented as four times. (d) CCN2 was compared with EGF (20 nM) and EGFR monoclonal antibody Erbitux (1 μM) for EGFR association in A549 cells under the conditions of detachment (top) for 24 h. The quantitative values were shown in the bottom. (e) Illustration of the sequential CT-domain deletion constructs or CT-domain construct of CCN2. (f) FACS analysis of cell membrane binding in rCCN2 (100 ng/ml), CT domain only recombinant protein (rCT, 100 ng/ml), and Erbitux (1 μM) treatment in A549 cells. After starvation, cells were treated as indicated and harvested to incubate with CCN2 antibody and secondary-FITC antibody. Cells were analyzed by flow cytometry. (g) Cys→Ala (C273A, C287A, and C292A) substitution of the CT domain constructs (top). Mutated CCN2 (C273A and C287A) lost its interaction with EGFR. WB-IP analysis of transfected wild-type/point mutation CCN2 (C273A, C287A and C292A) and vector (pcDNA3) expression plasmids in CL1-5 cells. Cells were transfected for 48 h and collected, and western blot and WB-IP were performed as indicated antibodies (bottom)