JCS03356 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. The characterization of anti-IQGAP antibodies. Purified MBP-tagged IQGAP-NRs, which includes each antigen, were subjected to SDS-PAGE, followed by immunoblot analysis with anti-IQGAP antibodies (upper three panels) and anti-MBP antibody (bottom panel). Three IQGAP antibodies recognized each IQGAP in a specific and a dose-dependent manner. The lower bands indicate the degradation products. The results represent three independent experiments.

• Supplemental Figure 2 -

  Fig. S2. The carboxyl region of IQGAP3 binds directly to activated Rac1 and Cdc42. Purified MBP-IQGAP3-C (749-1631aa), which includes GRD, was mixed with the affinity beads coated with the indicated GST-fused small GTPases. Bound IQGAP3-C was coeluted with GST-fused proteins by the addition of glutathione. The eluates were subjected to SDS-PAGE, followed by immunoblot analysis with anti-MBP antibody (top) and silver staining (bottom). The results shown are representative of three independent experiments.

• Supplemental Figure 3 -

  Fig. S3. Identification of actin as the IQGAP3 interacting molecule. Bovine brain cytosol was loaded onto beads coated with GST or GST-IQGAP3-N. The bound proteins were eluted by the buffer containing 500 mM NaCl. Aliquots of the eluates were resolved by SDS-PAGE, followed by silver staining (top) and immunoblot analysis with anti-actin antibody (bottom). The results shown are representative of three independent experiments.

• Supplemental Figure 4 -

  Fig. S4. The carboxyl region of IQGAP3 is important for neurite outgrowth in PC12 cells. (A) Schematic representation of the chimeric IQGAP1 (blue) and IQGAP3 (red) proteins. (B) Quantification of the total length of neurites in PC12 cells. The transfected cells were cultured in the presence of NGF. The total length of neurites was determined as described in Fig. 5C. Data are means ± s.d. of four independent experiments. n>150. Asterisks indicate the difference in the value of the control cells at P<0.01.

• Supplemental Figure 5 -

  Fig. S5. The effect of constitutively active Rac1 or Cdc42 on the morphology of PC12 cells without NGF. PC12 cells were transfected with EGFP-Rac1^V12 or EGFP-Cdc42^V12, followed by the incubation for 48 hours without NGF. The cells were labeled with anti-GFP antibody and Texas Red-phalloidin. The transfected cells were identified by anti-GFP antibody. The overexpression of Rac1^V12 or Cdc42^V12 affected themorphology of the cells, but failed to induce the typical neurites. The results shown are representative of four independent experiments. Bar, 20 μm.

• Supplemental Figure 6 -

  Fig. S6. Depletion of the IQGAP family in rat hepatoma cells. Total extracts from indicated siRNA-transfected MH1C1 cells were collected and subjected to immunoblot analysis with each anti-IQGAP antibody and anti-actin antibody. The expression level of each IQGAP was decreased specifically. The results shown are representative of five independent experiments.

• Supplemental Figure 7 -

  Fig. S7. The effects of the IQGAPs depletion on the growth cone morphology in hippocampal neuron. The neurons cotransfected with the indicated siRNA and Myc-GST were cultured on PDL alone. The cells were labeled with anti-Myc antibody (green), anti-unique β-tubulin (TUJ1) antibody (red), and Alexa Flour 647-phalloidin (blue) at DIV3. The transfected cells were identified by anti-Myc antibody. The results shown are representative of four independent experiments. Bar, 5 μm.