Figure 2. Direct interaction and... | Rockefeller University Press

Figure 2.

$Figure 2. Direct interaction and colocalization of MII with GEFs. (A) Specific interaction of endogenous MIIB with ectopically expressed Dbl family GEFs. PC12 cells were transfected with myc-tagged GEFs (right), or with ARNO or smgGDS GEF, which lacks a DH domain (left). Cell lysates were immunoprecipitated with anti-myc antibody followed by immunoblotting for MIIB (top). Expression of transfected genes was verified with anti-myc antibody (bottom). (B) Quantification of interactions between endogenous MIIA/MIIB and GEFs. Lysates (1 mg) from rat brain (E18), PC12, or NIH3T3 cells were immunoprecipitated with anti-MIIA or MIIB antibody followed by immunoblotting for the indicated GEFs. All of the immunoprecipitated proteins were loaded in each IP lane. A fraction (30 µg) of the same lysates for IP was loaded in each WCL lane. The percentage of each MII-bound GEF was calculated from dividing the relative densitometric values from the IP lane by those from the paired WCL lane. (C) Involvement of the DH–PH domain in interactions with MIIB. (Top) Myc-tagged GFP (control) or the DH domains from the various GEFs were expressed in PC12 cells. Lysates were immunoprecipitated with anti-myc antibody and immunoblotted for MIIB. (Bottom) Myc-tagged DH–PH domains from GEFT, collybistin, or Vav1 (positive control) were expressed and lysates were processed as described above. (D) Direct interaction of MII with DH–PH domains. Purified recombinant GST–DH–PH domains (2 µg) were incubated with MII (1 µg) from skeletal or cardiac muscle, then precipitated with glutathione agarose beads. Bound material was analyzed by immunoblotting for skeletal or cardiac MII. (E) Binding sensorgrams for the DH–PH domain and MII. His-tagged C-PIX (control, see Fig. 1 C) or DH–PH domain were immobilized on a Ni2+-NTA sensor chip and MII was passed over the immobilized proteins. Surface plasmon resonance was recorded and nonspecific binding of MII with C-PIX was subtracted from each DH–PH binding curve. (F) Colocalization of MIIs and GEFs. Swiss 3T3 cells were serum starved overnight and co-stained for βPIX (green) and MIIA/MIIB (red) (top), and for Trio (green) and MIIA/MIIB (red) (bottom). Enlarged images are shown on the right. Data are representative of three independent experiments. Bar, 10 µm.$

Direct interaction and colocalization of MII with GEFs. (A) Specific interaction of endogenous MIIB with ectopically expressed Dbl family GEFs. PC12 cells were transfected with myc-tagged GEFs (right), or with ARNO or smgGDS GEF, which lacks a DH domain (left). Cell lysates were immunoprecipitated with anti-myc antibody followed by immunoblotting for MIIB (top). Expression of transfected genes was verified with anti-myc antibody (bottom). (B) Quantification of interactions between endogenous MIIA/MIIB and GEFs. Lysates (1 mg) from rat brain (E18), PC12, or NIH3T3 cells were immunoprecipitated with anti-MIIA or MIIB antibody followed by immunoblotting for the indicated GEFs. All of the immunoprecipitated proteins were loaded in each IP lane. A fraction (30 µg) of the same lysates for IP was loaded in each WCL lane. The percentage of each MII-bound GEF was calculated from dividing the relative densitometric values from the IP lane by those from the paired WCL lane. (C) Involvement of the DH–PH domain in interactions with MIIB. (Top) Myc-tagged GFP (control) or the DH domains from the various GEFs were expressed in PC12 cells. Lysates were immunoprecipitated with anti-myc antibody and immunoblotted for MIIB. (Bottom) Myc-tagged DH–PH domains from GEFT, collybistin, or Vav1 (positive control) were expressed and lysates were processed as described above. (D) Direct interaction of MII with DH–PH domains. Purified recombinant GST–DH–PH domains (2 µg) were incubated with MII (1 µg) from skeletal or cardiac muscle, then precipitated with glutathione agarose beads. Bound material was analyzed by immunoblotting for skeletal or cardiac MII. (E) Binding sensorgrams for the DH–PH domain and MII. His-tagged C-PIX (control, see Fig. 1 C) or DH–PH domain were immobilized on a Ni²⁺-NTA sensor chip and MII was passed over the immobilized proteins. Surface plasmon resonance was recorded and nonspecific binding of MII with C-PIX was subtracted from each DH–PH binding curve. (F) Colocalization of MIIs and GEFs. Swiss 3T3 cells were serum starved overnight and co-stained for βPIX (green) and MIIA/MIIB (red) (top), and for Trio (green) and MIIA/MIIB (red) (bottom). Enlarged images are shown on the right. Data are representative of three independent experiments. Bar, 10 µm.