Figure 2. Cdc42 interacts directly with APC^222–653 in vitro.

(i) Cdc42-interacting APC clone from the brain cDNA library. The β-galactosidase activity of different bait and prey pairs in the Y2H screen. (a) Cdc42/N-WASP (positive control), (b) Cdc42/APC, (c) Cdc42/pACT2 (negative control) and (d) pAS2-1/N-WASP (negative control). (ii) Affinity pulldown assay. IRSp53 (positive control), GFP (negative control) and APC^222–653 were transcribed and translated using the TNT T7 coupled reticulocyte lysate systems kit in the presence of ³⁵S-methionine. The translated products were incubated with GST-tagged Cdc42V12 or Cdc42N17 or GST bound to glutathione-sepharose beads. The protein complexes were eluted by boiling and resolved by SDS-PAGE and detected by autoradiography. (a) Input for IRSp53, GFP and APC^222–653 respectively and (b) pulldowns using different affinity columns. First two lanes are Cdc42V12 pulldowns of ³⁵S-IRSp53 and ³⁵S-GFP respectively and the following three lanes are GST, Cdc42N17 and Cdc42V12 pulldowns of ³⁵S-APC^222–653. (iii) APC^222–653 interaction with Cdc42V12 as shown by AP-FRET. mRFP-APC^222–653 and GFP-Cd42V12 were coexpressed in CHO cells and AP-FRET analysis was carried out in the ROI (box). ROI line graphs showing the FRET assay results are shown below the image. Scale bar = 5µm. (iv) AP-FRET controls. AP-FRET analysis was carried out in the ROI (box). Line graphs showing the FRET assay results are shown on the right of the respective panels. (a) cell expressing cytosolic GFP-mRFP, a tandem fusion serving as positive control, (b) cell coexpressing cytosolic GFP and mRFP serving as negative control and (c) cell coexpressing mRFP-APC^222–653 and GFP-Cdc42N17. Scale bar = 5 µm. (v) Table shows the %FE and CC values of various APC constructs in the presence of (Cdc42V12, Cdc42N17, Rac1 or RhoA) along with positive and negative controls. Data is shown as ± SD, with three experiments and n = 7–10 cells for each experiment.