Results: 5

1.
Figure 2

Figure 2. BCAR3-induced cellular dissociation and BCAR3 membrane localization are independent of BCAR3-p130Cas complex formation. From: BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas.

(A) Polyclonal populations of MCF-7 cells stably transduced with an empty lentiviral vector, HA-BCAR3(WT) or HA-BCAR3(R743A) were photographed in culture at 40X magnification. (B) E-cadherin localization was assessed by immunofluorescent analysis of the same cell populations. Cells were counter-stained with DAPI and visualized at 60X magnification. (C) Stably transduced MCF-7 cells were probed with HA antibody to assess HA-tagged BCAR3 localization at 60X magnification. (D) Endogenous p130Cas localization in stably transduced MCF-7 cells was assessed with a p130Cas antibody at 60X magnification.

Pierre Vanden Borre, et al. Cell Signal. ;23(6):1030-1040.
2.
Figure 3

Figure 3. BCAR3-mediated p130Cas and HEF1 phosphoryation is independent of complex formation in stable polyclonal BALB/c-3T3 cells. From: BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas.

(A) Polyclonal populations of stably transduced BALB/c-3T3 cells expressing wildtype BCAR3, R743A BCAR3, wildtype NSP3 or chimeric NSP3/BCAR3 GEF were assessed for endogenous p130Cas, over-expressed HA-tagged proteins, endogenous Hef1 and tubulin by Western analysis. (B) HA immunoprecipitation of wildtype and R743A HA-BCAR3 from stably transduced polyclonal populations of BALB/c-3T3 cells. Immunoprecipitated lysates were probed with p130Cas antibody while WCL were probed with p130Cas and HA antibodies to detect endogenous p130Cas and over-expressed HA-tagged BCAR3, respectively. (C) λ phosphatase-treated HEF1 immunoprecipitates and whole cell lysates from BALB/c-3T3 cells were probed with HEF1 and HA antibodies to determine if the observed BCAR3-induced gel mobility shift represents a phosphorylated species. (D) NSP-mediated gel mobility shift of HEF1 was assessed by probing whole cell lysates from MCF-7 cells transiently co-transfected with plasmids encoding HEF1 and HA-tagged NSP family proteins with HEF1 antibody. The expression of exogenous NSP1, BCAR3 and NSP3 was confirmed by reactivity to anti-HA.

Pierre Vanden Borre, et al. Cell Signal. ;23(6):1030-1040.
3.
Figure 5

Figure 5. BCAR3-induced lamellipodia formation and motility in the absence of p130Cas. From: BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas.

(A) Western analysis of polyclonal populations of p130Cas-/- MEFs stably transduced with combinations of HA-BCAR3(WT), HA-BCAR3(R743A) and HA-p130Cas(WT). WCL were probed with antibodies to detect endogenous p130Cas, over-expressed HA-tagged proteins and tubulin. (B) As for panel A, a Western analysis of endogenous HEF1 expression and phosphorylation. (C) Phase contrast images of p130Cas-/- MEFs and polyclonal populations of stably transduced p130Cas-/- MEFs (40X magnification). p130Cas-/- MEFs and p130Cas-/- MEFs expressing either wildtype or R743A BCAR3 are shown in the left column and p130Cas-/- MEFs reconstituted with wildtype p130Cas and also expressing wildtype or R743A BCAR3 are shown in the right column. (D) Quantification of BCAR3-induced lamellipodia formation in p130Cas-/- MEFs and stably transduced polyclonal populations of p130Cas-/- MEFs. (E) Mean rates of migration of p130Cas-/- MEFs were determined by measuring the distance traveled by cells over a 24 hour period following the scratching of a confluent cell monolayer. Significance was determined by one-way ANOVA (asterisk p < 0.05 relative to p130Cas-/-; triangle p < 0.05 relative to p130Cas-/- + BCAR3(WT)).

Pierre Vanden Borre, et al. Cell Signal. ;23(6):1030-1040.
4.
Figure 4

Figure 4. BCAR3-induced membrane lamellipodia formation is independent of association with p130Cas in BALB/c-3T3 cells, while BCAR3-induced motility is not. From: BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas.

(A) Phase contrast images of polyclonal populations of BALB/c-3T3 cells stably transduced with either an empty lentiviral vector, wildtype BCAR3 or R743A BCAR3 at 40X magnification. (B) BALB/c-3T3 cells and polyclonal populations of BALB/c-3T3 cells stably transduced with wildtype BCAR3 or R743A BCAR3 were stained with phalloidin (red) and DAPI (blue) to visualize actin stress fibers and nuclei, respectively (40x magnification). (C) BALB/c-3T3 cells and BALB/c-3T3 cells stably transduced with either wildtype or R743A BCAR3 were probed with anti-HA primary and Alexa Fluor 647 secondary antibodies to determine BCAR3 localization at 60X magnification. (D) Quantification of BCAR3-induced lamellipodia formation in BALB/c-3T3 and stably transduced populations of BALB/c-3T3 cells. Error bars depict the propagated error of the mean and significance was determined by one-way ANOVA (asterisk, p < 0.05 relative to BALB/c-3T3). (E) Mean rates of migration of BALB/c-3T3 cells were determined by measuring the distance traveled by cells over a 16 hour period following the scratching of a confluent cell monolayer. Significance was determined by one-way ANOVA (p < 0.05).

Pierre Vanden Borre, et al. Cell Signal. ;23(6):1030-1040.
5.
Figure 1

Figure 1. BCAR3-mediated anti-estrogen resistance and Rac activation are independent of BCAR3-p130Cas complex formation. From: BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas.

(A) A clonal MCF-7 cell line stably transfected with HA-tagged BCAR3 (II-6 cells) or polyclonal MCF-7 cell populations stably transduced with either a control lentivirus or wildtype or R743A HA-tagged BCAR3-expressing lentivirus were immunoprecipitated with HA antibody and probed with p130Cas antibody (left panel). Whole cell lysates (WCL) were probed with HA antibody to demonstrate expression of the exogenous BCAR3 and with p130Cas antibody to assess phosphorylation of endogenous p130Cas as determined by a shift in p130Cas gel mobility (right panel). (B) MCF-7 cells stably transfected with HA-tagged BCAR3 were transiently transfected with wildtype or L791D p130Cas, followed by immunoprecipitation with BCAR3 antibody and probing with an HA antibody. WCL from these two cell types as well as MCF-7 cells transiently transfected with wildtype p130Cas were probed with HA antibody to examine the expression and electrophoretic mobility of exogenous BCAR3 and p130Cas. (C) Following treatment with 100nM fulvestrant (ICI 182,780), parental MCF-7 cells or polyclonal populations stably transduced with empty, wildtype BCAR3, R743A BCAR3, NSP3 or NSP3/BCAR3 GEF-expressing lentivirus were assessed for cell growth. Experiments were performed three times. Asterisks indicate significant differences in cell growth (p < 0.05). (D) The PAGE migration of p130Cas was examined in WCL of either wildtype MCF-7 cells or polyclonal MCF-7 cells stably transduced with control, wildtype or R743A BCAR3 or wildtype or chimeric NSP3/BCAR3 GEF lentivirus. Expression of the NSP family members was confirmed by probing with HA antibody. (E) GST-PAK1 pulldown assay to detect GTP-bound Rac in MCF-7 cells transiently transfected with empty vector, HA-BCAR3(WT), HA-BCAR3(R743A) or HA-BCAR3(ΔGEF). WCL were probed for total Rac and HA. The pulldown assay was performed in triplicate.

Pierre Vanden Borre, et al. Cell Signal. ;23(6):1030-1040.

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