Figure 2: PTEN inhibits the CAV1/β-catenin immunocomplex.

(a) Interactome of PTEN and β-catenin (BCAT) as determined by the in silico Ingenuity Pathway Analysis (IPA). The Venn diagram reveals 63 common members. Interaction of CAV1 with β-catenin (BCAT; b) and PTEN (c) in Rosi human melanoma cells. Extracts from ∼1.5 × 10⁷ cells (corresponding to about 1.5 mg) were immunoprecipitated with control IgG, anti-BCAT, anti-CAV1 or anti-PTEN antibodies. Immune complexes were resolved by SDS–PAGE and blotted with antibodies to BCAT, CAV1 and PTEN. Total protein input corresponds to 2% of the total protein used for immunoprecipitation. One-tenth of the IP sample was loaded to detect BCAT following IP with BCAT antibodies (and similarly for CAV1 and PTEN). This allowed getting a reasonable intensity for the corresponding signals. For the other lanes, the entire IP samples were loaded. (d) GST pulldown using β-catenin–GST fusion Sepharose beads and whole-cell protein lysates (500 μg) from Hs944T cell transfection with GFP or PTEN. Pellet and supernatant fractions were immunoblotted for various antibodies. (e) CAV1-BCAT immunocomplex in GFP transiently transfected Hs944T cells. When transfected with PTEN, the proportion of β-catenin in the immunocomplex is dramatically reduced. Total protein input is shown. For b–e, experiments were performed three times. (f–k) Immunofluorescence of transiently transfected Hs944T human melanoma cell line with CMV::GFP and CMV::CAV1-RFP (CAV1) expression vector. β-Catenin staining (f,i), GFP (g), CAV1 (j) and merged (h,k). Scale bar, 25 μm. Immunofluorescence experiments for Hs944T cells were performed three times with similar results. (l–t) Immunofluorescence of mouse carcinoma submandibular gland (CSG) cells transfected with siRNA directed against either negative control (siScr), PTEN (siPTEN) and CAV1 (siCAV1) stained for BCAT (l,o,r), respectively. Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (m,p,s). Merge (n,q,t). Scale bar, 25 μm for all panels. Immunofluorescence experiments for the CSG cells were performed two times with similar results.