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1.
FIGURE 2.

FIGURE 2. From: BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling.

Two distinct pools of Cas exist in BT549 cells. A, Cas and c-Src are present in a molecular complex. 400 μg of BT549 cell lysate were incubated with c-Src monoclonal antibody 2-17 (lane 2) or isotype-matched monoclonal antibody HA.11 as a control (lane 3). Immune complexes were collected and immunoblotted with the indicated antibodies. For comparison, 20 μg of cell lysate were immunoblotted (lane 1). The vertical separation indicates different exposure times between lysate and immunoprecipitation (IP) lanes. B, Cas and BCAR3 are present in a molecular complex. BCAR3 or control (preimmune serum) immune complexes were generated as described above from 350 μg of BT549 cell lysate, collected on protein A-agarose, and immunoblotted with the indicated antibodies (lanes 2 and 3). For comparison, 30 μg of cell lysate were immunoblotted (lane 1).

Natasha R. Schuh, et al. J Biol Chem. 2010 January 22;285(4):2309-2317.
2.
FIGURE 1.

FIGURE 1. From: BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling.

BCAR3 augments Cas-mediated enhancement of c-Src activity. A, co-overexpression of Cas and BCAR3 enhances Cas-dependent c-Src activity. COS-1 green monkey kidney cells were transfected with plasmids encoding wild type c-Src, FLAG-cortactin, pRK5 vector (V), Myc-Cas (C), and/or FLAG-BCAR3. FLAG immune complexes were generated from cell lysate 24 h post-transfection and immunoblotted with Tyr(P) and FLAG-specific antibodies (top two panels). Whole cell lysates were immunoblotted with Myc and FLAG antibodies (bottom two panels). IP, immunoprecipitation. B, BCAR3 requires Cas/c-Src binding to augment c-Src activity. COS-1 green monkey kidney cells were transfected with plasmids encoding wild type c-Src, FLAG-paxillin, pRK5 vector (V), Myc-Cas (C), Myc-Cas-TM (TM), and/or FLAG-BCAR3. Cells were lysed 24 h post-transfection, and proteins were analyzed as described above. Data shown are representative of 2–4 independent experiments.

Natasha R. Schuh, et al. J Biol Chem. 2010 January 22;285(4):2309-2317.
3.
FIGURE 7.

FIGURE 7. From: BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling.

Proposed mechanism of BCAR3-mediated enhancement of c-Src activation, cell spreading, and adhesion. Step 1, cytosolically localized Cas is recruited to membrane-proximal regions by BCAR3 through an indirect or direct interaction with BCAR3. Once at the membrane (step 2), Cas interacts with and activates c-Src. c-Src activation results in the phosphorylation of Cas, which leads to the recruitment of substrate domain binding partners such as Crk (step 3). Step 4, Cas/Crk interactions activate downstream signaling pathways important for cell spreading, migration, and invasion. BCAR3-dependent and BCAR3-independent c-Src signaling can also activate other signaling pathways important for cell proliferation and survival in the presence of antiestrogens. Step 5, BCAR3 itself activates an unidentified c-Src-independent pathway important for cell spreading.

Natasha R. Schuh, et al. J Biol Chem. 2010 January 22;285(4):2309-2317.
4.
FIGURE 6.

FIGURE 6. From: BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling.

BCAR3 regulates both c-Src-dependent and -independent signaling pathways in response to adhesion and cell spreading. A, BT549 cells were transfected with nontargeting or BCAR3-specific siRNA duplexes and grown for 72 h. Cells were left in suspension for 90 min in the presence of DMSO or 10 μm SU6656 and then plated onto fibronectin-coated coverslips in complete growth media with 10% fetal bovine serum for the indicated lengths of time in normal growth media with or without SU6656. B, mean cell area was calculated from four independent experiments. Two-tailed Student's t tests were conducted for comparison between the various sample sets. Bars indicate standard deviation. *, #, and ⋀ indicate significant difference from the mean versus control DMSO, control SU6656, and siBCAR3 DMSO, respectively, at ≥95% confidence interval.

Natasha R. Schuh, et al. J Biol Chem. 2010 January 22;285(4):2309-2317.
5.
FIGURE 3.

FIGURE 3. From: BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling.

Depletion of BCAR3 in BT549 breast cancer cells disrupts c-Src activation, Cas phosphorylation, and Cas/c-Src interactions. A, BT549 cells depleted for BCAR3 have decreased c-Src activity. BT549 breast cancer cells were transfected with nontargeting or BCAR3-specific siRNA duplexes and grown for 48 h. Whole cell lysate was immunoblotted with the indicated antibodies. The mean relative Tyr(P)-419 (α-pY419) phosphorylation was determined from four independent experiments. B, BT549 cells depleted for BCAR3 have decreased Cas phosphorylation. Cas immune complexes were generated from 72-h siRNA-treated BT549 cells and immunoblotted with antibodies specific to Tyr(P) and Cas (top two panels). Cell lysate was immunoblotted for BCAR3 to verify loss of BCAR3 expression (bottom panel). The mean relative Cas phosphorylation was determined from four independent experiments. C, BT549 cells depleted for BCAR3 have decreased Cas/c-Src interactions. c-Src immune complexes were generated from 72-h siRNA-treated BT549 cells and immunoblotted with Cas and c-Src antibodies (top panel). Whole cell lysate was also immunoblotted with BCAR3 antibodies (bottom panel). The mean Cas levels in Cas-c-Src immune complexes were determined from five independent experiments. Two-tailed Student's t tests were conducted for comparison between siControl and siBCAR3 samples for A–C. Bars indicate standard deviation; asterisk indicates significant difference from the mean at ≥95% confidence interval. Within all panels, exposure time is the same; the white vertical lines denote noncontiguous sample lanes.

Natasha R. Schuh, et al. J Biol Chem. 2010 January 22;285(4):2309-2317.
6.
FIGURE 4.

FIGURE 4. From: BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling.

BCAR3 overexpression in MCF7 breast cancer cells increases c-Src activity and Cas phosphorylation. A, Dox-regulated expression of BCAR3 in Tet-Off MCF7 cells. MCF7 cells overexpressing BCAR3 under control of a tetracycline-responsive promoter were grown in the presence or absence of 2 μg/ml Dox for 72 h. Whole cell lysate was immunoblotted with antibodies specific to BCAR3 and β-tubulin. B, BCAR3 overexpressing cells exhibit increased c-Src activation. c-Src immune complexes were generated from MCF7 cells grown in the presence or absence of 2 μg/ml Dox for 72 h and immunoblotted with antibodies specific for Tyr(P)-419 and total c-Src. Tyr(P)-419 was increased by an average of 1.9 ± 0.43-fold in three-independent experiments (p = 0.02). The white vertical line denotes noncontiguous sample lanes; the exposure times for the +/− Dox samples in each immunoblot is the same. C, BCAR3-dependent Cas phosphorylation is mediated by Src family kinases. MCF7-B6 cells grown in the presence or absence of Dox for 48 h were treated with vehicle (DMSO) or 10 μm SU6656 and grown for an additional 24 h. Cas immune complexes were generated from lysate and immunoblotted with antibodies specific to Tyr(P) and Cas (top two panels). Whole cell lysate was also immunoblotted with BCAR3 antibodies (bottom panel). Cas phosphorylation increased by an average of 2.9 ± 0.64-fold (p = 0.009) in the presence of overexpressed BCAR3. Data shown are representative of four independent experiments. D, BCAR3 expression regulates Cas/c-Src interactions. c-Src immune complexes were generated from MCF7 cells grown in the presence or absence of 2 μg/ml Dox for 72 h and immunoblotted with Cas- and c-Src-specific antibodies.

Natasha R. Schuh, et al. J Biol Chem. 2010 January 22;285(4):2309-2317.
7.
FIGURE 5.

FIGURE 5. From: BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling.

Loss of BCAR3 expression disrupts cell spreading and adhesion-mediated Cas phosphorylation. A and B, BT549 cells depleted for BCAR3 exhibit decreased cell spreading on fibronectin. BT549 cells were transfected with nontargeting or BCAR3-specific siRNA duplexes and grown for 72 h. Cells were left in suspension for 90 min and then plated on fibronectin-coated coverslips in complete growth media with 10% fetal bovine serum for the indicated lengths of time in normal growth media. A coverslip from cycling, adherent cells was also examined. Mean cell area from three independent experiments was determined by using ImageJ software. Bars indicate standard deviation; asterisk indicates significant difference from the mean at ≥95% confidence interval. C, BT549 cells depleted for BCAR3 exhibit decreased adhesion-mediated Cas phosphorylation. BT549 cells were transfected and treated as described above. Cas immune complexes were generated from lysate and immunoblotted with Tyr(P) and Cas antibodies (top two panels). Whole cell lysate was also immunoblotted with antibodies specific to BCAR3 (bottom panel). Data shown are representative of four independent experiments. Within all panels, exposure time is the same; the white vertical line denotes noncontiguous sample lanes. IP, immunoprecipitation.

Natasha R. Schuh, et al. J Biol Chem. 2010 January 22;285(4):2309-2317.

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