IQGAP1 modulates activation of B-Raf

Ren et al. 10.1073/pnas.0611308104.

Supporting Information

Files in this Data Supplement:

SI Figure 6
SI Figure 7
SI Figure 8

Fig. 6. B-Raf interacts with the N-terminal half of IQGAP1. (A) Schematic representation of IQGAP1 constructs. Full-length (WT), N-terminal (N), and C-terminal (C) halves of IQGAP1 are depicted. (B) [³⁵S]methionine-labeled wild-type IQGAP1, IQGAP1-N, and IQGAP1-C were incubated with equal amounts of GST-B-Raf on glutathione-Sepharose beads. GST alone was used as control. Complexes were washed, resolved by SDS/PAGE, and processed by autoradiography. Aliquots of [³⁵S]methionine-labeled TNT products (equivalent to 10% of the amount that was subjected to pull-down) were resolved by SDS/PAGE (Input). (C) myc-IQGAP1 (WT), myc-IQGAP1-N (N), or myc-IQGAP1-C (C) was cotransfected with HA-B-Raf into HEK 293H cells. After lysis, equal amounts of protein were immunoprecipitated with anti-myc monoclonal antibody or incubated with nonimmune mouse serum (NIMS). Both immune complexes and unfractionated lysate (Lys) were resolved by SDS/PAGE, transferred to PVDF, and probed with anti-myc monoclonal (for IQGAP1) and anti-HA (for B-Raf) polyclonal antibodies. The position of migration of molecular weight markers is depicted on the left side of the figure. Data are representative of two independent experiments.

Fig. 7. Identification of the amino acids residues of IQGAP1 necessary for B-Raf binding. (A) Schematic representation, nomenclature, and amino acids boundaries of the fragments of human IQGAP1 used in this figure. (B) GST-B-Raf or GST alone immobilized on glutathione-Sepharose beads was incubated with equal amounts of [³⁵S]methionine-labeled IQGAP1-N (N), IQGAP1-C (C), IQGAP1-N1 (N1), IQGAP1-N2 (N2), or IQGAP1-(763-964) (763-964). Complexes were washed, resolved by 10% SDS/PAGE, and processed by autoradiography. Input is equivalent to 10% of the amount that was subjected to pull-down. Data are representative of two independent experiments. (C) GST-Cdc42V12, GST-B-Raf, or GST alone immobilized on glutathione-Sepharose beads was incubated with equal amounts of [³⁵S]methionine-labeled IQGAP1-(763-964) (763-964), and IQGAP1-C (C). Complexes were washed, resolved by 12% SDS/PAGE, and processed by autoradiography. Data are representative of two independent experiments.

Fig. 8. IQGAP1DB-Raf does not bind B-Raf. GST-B-Raf or GST alone immobilized on glutathione-Sepharose beads was incubated with equal amounts of [³⁵S]methionine-labeled wild-type (WT) IQGAP1 or IQGAP1ΔB-Raf (ΔRaf). Complexes were processed by SDS/PAGE and autoradiography. Input is ~10% of the amount that was subjected to pull-down. Data are representative of two independent experimental determinations.

• Early Edition
• Archives
• Online Submission

• Feature Articles

  (Expanded research articles of exceptional breadth)

• Commentaries

  (Expert commentary on leading research)

• Letters

  (Online-only letters to the editor)

• Inaugural Articles

  (Articles by recently elected NAS members)

• PNAS Profiles

  (Biographical profiles of Academy members)

• Sustainability Science

  (Interactions of human and environmental systems)

• Collected Papers

  (Editorials, Perspectives, Reviews, Colloquia, etc.)

• Special Features

  (Solicited articles on exceptional research topics)

• Sackler Colloquia

  (Scientific reports held under NAS auspices)