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. 2007 Jan 25;26(3):647–656. doi: 10.1038/sj.emboj.7601534

Figure 2.

Figure 2

Involvement of Cbl in invasion. (A) AMAP1 interacts with Cbl via CIN85. HEK293T cells were transfected with GST-AMAP1, Xpress-CIN85 and Cbl-b, as indicated. GST-AMAP2 was included as a control. GST proteins were pulled down from 200 μg of cell lysates and subjected to immunoblotting analysis using antibodies, as indicated. Total, 5 μg of total cell lysates. (B) EGF-dependent interaction of AMAP1 and Cbl in MDA-MB-231 cells. Cells were stimulated with or without 100 ng/ml EGF for 10 min. Cell lysates (600 μg) were then subjected to immunoprecipitation with an anti-AMAP1 antibody, followed by immunoblotting using antibodies, as indicated. Total, 10 μg of total cell lysates. (C, D) Involvement of Cbl and its binding to CIN85 in Matrigel chemoinvasion. MDA-MB-231 cells were treated with siRNAs for c-Cbl and/or Cbl-b (C), or treated with Cbl-b siRNA and transfected with rescue constructs of wild-type Cbl-b (Cbl/resWT) and its R911A mutant (resR911A) (D). Cells were then cultured on Biocoat Matrigel chambers to measure Matrigel chemoinvasion activity. siRNA with an irrelevant sequence (irr) was included as a control. Data collection and presentation are the same as in Figure 1C (lower panel). Results represent the mean±s.e. of three independent experiments. The upper panels show protein expression in these cells by immunoblotting.