JCS025536 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) Subcellular localization of total and dephospho-β-catenin in colon carcinoma cell lines. (B) Relative E-cadherin protein levels in cell lines shown in A. 20 µg of total cellular protein was separated on SDS-PAGE, blotted and probed with an anti-E-cadherin antibody. Western blot signals were quantified using a luminoscan analyzer. Equal loading was confirmed using β-actin detection, levels of which varied less than 25%.

• Supplemental Figure S2 -

  Fig. S2. (A) Membrane localization of dephospho-β-catenin does not coincide with membrane localization of cadherins. Cells were stimulated as in A and stained for dephospho-β-catenin and cadherins using a pan-cadherin antiserum. As a reference, E-cadherin^+/+ cells are used. (B) Plasma membrane recruitment of dephospho-β-catenin is independent of ongoing transcription. E-cadherin^−/− cells were analyzed as in A in the presence or absence of the transcription inhibitor actinomycin D at 4 µg/ml. No significant difference in plasma membrane localization of dephospho-β-catenin was observed. P values according to Fisher’s exact tests.

• Supplemental Figure S3 -

  Fig. S3. Generation of anti-APC antisera. (A) Domain structure of APC. Coiled coil and basic regions are shown as dark grey boxes at the N- and C-terminal ends of APC, respectively. Armadillo repeats (light blue), and β-catenin (yellow and green) and axin (red) binding regions are also depicted. The domains (amino acids indicated) used to make GST fusion proteins are shown above the sequence. (B-D) Western blot analysis. In (B) a blot is shown of overexpressed eCFP, and eCFP-tagged APC-A, -B and -C in COS-1 cells. Proteins were detected with anti-GFP antibodies. (C) Blot of eCFP-APC-A (left panel) or eCFP-APC-B (right panel), overexpressed in COS-1 cells, and detected with five rat monoclonals against APC-A or with five monoclonals against APC-B. Note that only some antibodies recognize the eCFP-tagged proteins. Strips incubated with 13F7 and 3E7 are indicated. (D) Blots are shown with indicated cell lysates. Of the four cell lines, used only HCT116 expresses full-length APC (indicated as wt). The residues at which APC is truncated in the other cell lines are indicated. Note that 3E7 only recognizes full-length APC (and degradation products), whereas 13F7 recognizes both full-length and truncated forms of APC (E,F) on immunofluorescence. (E) SW480 and MDCK cells were fixed and stained with the indicated antibodies (IN, polyclonal rabbit antibody donated by Inke Nathke). APC is found clustered in peripheral domains (indicated by arows). Note that 3E7 fails to stain such clusters in SW480 cells. (F) Double labelling of 3E7 with anti-tubulin antibodies. APC is concentrated at the ends of a subset of microtubules (arrows). The staining patterns obtained with 3E7 and 13F7 are comparable with that obtained with the IN antibody and resemble published patterns.

• Supplemental Figure S4 -

  Fig. S4. Low-power photomicrograph of consecutive sections of normal human small intestinal crypt-villus axis stained for (A) total β-catenin and (B) dephospho-β-catenin. Labeling for total β-catenin replicates dephospho-β-catenin staining, demonstrating enhanced membranous labelling at the level of the crypt compartment over the villus compartment.

• Supplemental Figure S5 -

  Fig. S5. β-catenin antibody 06-734 predominantly recognizes dephosphorylated β-catenin. HEK 293T cell lysates were prepared in standard 1% NP-40 lysis buffer, clarified and subsequently used for immunoprecipitation with 1 µg anti-total-β-catenin (C19220, Transduction laboratories). Phosphatase treatment on the immunoprecipitate was carried out with 200 U lambda phosphatase (λPPase) at 37°C for 30 minutes. Samples were eluted in sample buffer and loaded on a gel and blotted for 8E7 and 06-734 at 1:500 and 1:1000 dilution, respectively. An 8E7 crossreacting band (*) is indicated.