JCS02864 Supplementary Material

Files in this Data Supplement:

• Supplemental Table - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Non-binding controls. (A) Compartment FRAP analysis of mYFP- b-catenin in the absence or presence of CFP alone or of the indicated CFP fusion proteins. KID is a domain of the transcription factor CREB; the NIPP1 (nuclear inhibitor of protein phosphatase 1) expression plasmid was generously provided by L. Trinkle-Mulcahy. (B) Compartment FRAP analysis of mYFP- b-cateninDarm2-5 and mYFP- b-catenin wt in the absence or presence of mCFP-BCL9v. (C) Point bleach analysis of mYFP- b-cateninDarm2-5 and mYFP- b-catenin wt in the absence or presence of mCFP-axin or mCFP-APC.

• Supplemental Figure 2 -

  Fig. S2. E-cadherin slows down b-catenin in the cytoplasm. (A) Live cell image of mYFP- b-catenin in cells co-transfected with untagged E-cadherin (bar, 10 µm). (B) Compartment bleach analysis of mYFP- b-catenin shuttling in the absence or presence of E-cadherin. (C) Point bleach analysis of mYFP- b-catenin mobility in the cytoplasm in the absence or presence of E-cadherin. Cell-cell contacts were not included in the analysis.

• Supplemental Figure 3 -

  Fig. S3. Integrity of the expressed proteins. (A) Lysates from 293T cells expressing mYFP-tagged b-catenin wt and b-catenin deletions separated on an 8% SDS polyacrylamide gel and detected with anti-GFP antibody (7.1/13.1, Roche).  (B) Lysate from 293T cells expressing mYFP-tagged APC separated on a 4% SDS polyacrylamide gel detected with anti GFP antibody (left lane); as a control for the expected size of the protein, a lysate of untransfected cells was separated on the same blot and detected with mouse anti-APC antibody Ali (generously provided by I. Näthke) (right lane). (C) Lysates from 293T cells expressing the indicated mYFP-tagged proteins separated on an 8% SDS polyacrylamide gel and detected with anti-GFP antibody.

• Supplemental Figure 4 -

  Fig. S4. Effects of untagged b-catenin interactors. (A,C,E,G) Compartment bleach analysis of b-catenin in the absence or presence of mCFP-tagged or untagged TCF4/Lef1 (A), BCL9v/Pyg2 (C), APC (E) and axin/axin2 (G).  (B,D,F,H) Point bleach analysis of b-catenin mobility in the absence or presence of mCFP-tagged or untagged TCF4/Lef1 (B), BCL9v/Pyg2 (D), APC (F) and axin/axin2 (H) in the indicated compartment.

• Supplemental Figure 5 -

  Fig. S5. New protein synthesis is not relevant for fluorescence recovery. Mean total cell fluorescence of 20 cells expressing mYFP-tagged b-catenin during compartment bleach experiments; total cell fluorescence drops down to approx. 45% of the initial value immediately after the bleach and does not recover to more than 48% within the 15 minutes monitoring time, indicating that the contribution of newly synthesized protein to fluorescence recovery is negligible.

• Supplemental Figure 6 -

  Fig. S6. Effects of CFP versus YFP tags. (A) Half recovery times of mCFP- b-catenin and mYFP- b-catenin show similar recovery kinetics of both proteins. (Recovery curves are not depicted because of lower bleach efficiencies of mCFP- b-catenin due to a weaker laser line, which resulted in different starting points after the bleach.) (B) Compartment bleach analysis of mYFP-mYFP compared to YFP-CFP; mYFP-b-catenin is shown for comparison.

• Supplemental Figure 7 -

  Fig. S7. Lack of saturation of the importin/exportin system. Plot of nucleo-cytoplasmic ratios (logarithmic scale) versus total cell fluorescence of HEK293T cells expressing proteins with or without strong NES/NLS sequences. Values were determined from confocal images of live cells. Each icon represents a single cell. Cells were imaged with constant exposure parameters to allow comparison of the relative protein expression levels. The boxed area shows the typical expression range in cells used for FRAP experiments. Note that over the whole range of expression levels, the nucleo-cytoplasmic distribution of all proteins remains constant, indicating that the endogenous importin/exportin system is not overloaded in our assays.