JCS029470 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) N3 forms a dimer. Purified N3 migrates as a band with an apparent molecular mass of about 65 kD on a native PAGE gel. The same band reacts with antibodies against the His-tag attached to N3. The predicted molecular mass of N3 is ca. 30 kD. (B) N2 does not bind to endogenous N-APC. Equal amount of proteins from cell lysates of SW480 cells transfected with N2 or GFP were immunoprecipitated with a monoclonal anti-N-APC antibody. Total lysate and proteins bound to N-APC were probed with an anti-GFP antibody. Unlike N3, N2 does not co-precipitate with endogenous N-APC.

• Supplemental Figure S2 -

  Fig. S2. 14-3-3 overlay experiments. SW480 cells were transfected with different GFP-tagged N-APC fragments and incubated with medium containing 1% (to prevent phosphorylation) or 10% serum. Cell lysates were immunoprecipitated with a monoclonal anti-GFP antibody and precipitated proteins were subjected to 14-3-3 protein overlay using Digoxygenin-conjugated 14-3-3 (top panel). The same blot was also probed with anti-GFP antibodies (bottom panel) to show precipitated proteins. The fragment called N580 spans residues 1-580, so encompasses N1 plus part of N2.

• Supplemental Figure S3 -

  Fig. S3. The cluster types formed by endogenous full-length APC in MDCK cells stably expressing N3 was scored as in Fig. 4B in at least 100 cells from three independent experiments.

• Supplemental Figure S4 -

  Fig. S4. Cluster formation in PTK2 cells. (a) Cluster types were scored as in Fig. 4B in PTK2 cells after transient transfection with full-length GFP-APC plus N3-GST or GST. The presence of N3 led to a decrease in the clustering of APC. (b) Cluster types in cells transiently transfected with GFP-APCΔN3 or GFP-APC were scored as in Fig. 4B. Clusters formed by APCΔN3 tended to be more tightly clustered and less dispersed. In each case, 100 cells were randomly selected from three independent experiments.

• Movie 1
• Movie 2
• Movie 3 -

  Movies 1-3. Fluorescence recovery after photobleaching of GPF-APC clusters. Three movies are included in the supplemental data showing FRAP experiments on clusters formed by APC-GFP (Movie 1 and 2) or GFP-ΔN3APC (Movie 3). These recordings illustrate that clusters rearrange and change shape and/or move more quickly than the bleached spot recovers so that recovery of a bleached spot within a cluster cannot be quantified accurately. This makes it impossible to quantify any molecular dynamics to measure APC exchange within the clusters using this technique.