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Figure 2


Fig. 2. Interaction between AMER1 and APC. (A) Co-immunoprecipitation of wild-type APC (APC) and endogenous mutant APC (APCmut) with Flag-tagged AMER1 after transient transfections of SW480 cells, as indicated. Western blottings were performed using anti-APC Ab1 (for APCmut) and Ab2 (for APC), and anti-Flag antibodies. The double band for Flag-AMER1 is observed in some but not all experiments (cf. B) and might result from incomplete denaturation of the protein in the gel sample buffer. (B) Co-immunoprecipitation of endogenous AMER1 and Flag-tagged AMER1 with EGFP-tagged APC-ARM after transient transfections of 293T cells, as indicated. Western blottings were performed using anti-AMER1 or anti-GFP antibodies. (C) Co-immunoprecipitation of endogenous AMER1 with APC from lysates of nontransfected 293T and SW480 cells. Immunoprecipitations were performed with anti-GFP antibody as a control or with anti-APC antibody Ali followed by western blotting using the anti-AMER1 antibody or Ali. (D) Co-immunoprecipitation of EGFP-tagged APC-ARM or APC-ARMN507K with Flag-tagged AMER1 or AMER1(short) after transient transfections of 293T cells as indicated. Western blottings were performed using anti-GFP or anti-Flag antibodies. (E) Co-immunoprecipitation of Flag-tagged APC-ARM with EGFP-tagged AMER1 and AMER1 deletion fragments after transient transfections of 293T cells as indicated. Western blottings were performed using anti-Flag or anti-GFP antibodies. The bottom blot shows levels of the Flag-FKBP8 control protein in lysates of 293T cells after co-expression with the indicated AMER1 constructs. The scheme below shows the structure of the deletion mutants of AMER1 and quantification from separate experiments of the fold change of APC-ARM or FKBP8 protein levels (as a control) after co-transfection with the indicated AMER1 constructs, relative to EGFP transfection. (F) Western blotting for APC (antibody Ali) in stable clones of MDCK cells expressing EGFP (EGFP#1, EGFP#2) or EGFP-tagged AMER1 (EGFP-AMER1#1, EGFP-AMER1#2). (G) Western blotting for APC (antibody Ali), AMER1, Pan-cadherin and FKBP8 from lysates of 293T cells transiently transfected with siRNA oligonucleotides against GFP (as a control), or two different siRNA oligonucleotides against AMER1 (siAMER1a, c). The numbers below the lanes indicate relative protein levels normalized to Pan-cadherin, with siGFP controls set to 100%.





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