JCS011320 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Genomic localization and expression analysis of Amer1. (A) Schematic representation of the human Amer1 gene locus. Exon/intron structures and splicing patterns were deduced by matching the Amer1 coding sequence and the FLJ39827 cDNA to the genomic database (NCBI, clone RP11-403E24). The dashed lines at exons indicate that the boundaries could not be deduced because of missing cDNA information. The mRNA sequences of Amer1 and FLJ39827 are indicated by thick lines and the splicing pattern by thin lines. As the complete transcript of Amer1 has not been cloned, dashed lines 5′ of the ATG and 3′ of the stop codon are hypothetical. The common ATG (start) codon of Amer1 and FLJ39827 and the different stop codons are indicated. (B) RT-PCR analysis of Amer1 and APC expression in different human tissues. RT-PCR for GAPDH was used for normalization. RT-PCR analysis was performed using total cellular RNA isolated with peqGold Trifast reagent (Peqlab, Erlangen, Germany). Single-stranded cDNA was synthesized from 1 μg total cellular RNA or 1 μg RNA from a Human Total RNA Master Panel (Clontech) using Superscript II reverse transcriptase (Invitrogen GmbH, Karlsruhe, Germany). RT-PCR was performed using the following sense and antisense primers: Amer1 (5′-Cactcaaaacctccctgagctgc-3′, 5′-catagcagcaggtggaggtcgag-3 at 62°C annealing temperature, 30 cycles); APC (5′-aagttgcggccgctgggaacc-aaggtggaaatggtg-3′, 5′-aagtcgcggccgcctattcaacaggagctggc-attg-3′ at 55°C annealing temperature; 28 cycles) and GAPDH (5′-CCACCCATGGCAAATTCCATGGCA-3′, and 5′- TCTAGACGGCAGGTCAGGTC-CACC-3′ at 58°C annealing temperature; 22 cycles). (C) Western blotting of Amer1 using the anti-Amer1 mouse monoclonal antibody. Left panels, western blot of 293T cells transiently transfected with Flag-tagged Amer1 or siRNA oligonucleotides as indicated. Note that the antibody detects a band of about 190 kDa, for both endogenous and exogenous Amer, which is diminished after specific siRNA treatment against Amer1. Right panels, anti-Amer1 western blots of various human cell lines.

• Supplemental Figure S2 -

  Fig. S2. Amer2 genomic localization, sequence comparison with Amer1 and interaction with APC. (A) Schematic representation of the human Amer2 gene locus. Exon/intron structures and splicing patterns were deduced by matching the Amer2 cDNA sequences of transcript variant 1 and 2 (NCBI accession number NM_152704 and NM_199138, respectively) to the genomic database (Homo sapiens chromosome 13 genomic contig, NCBI accession number NT_024524.13). The dashed lines at exons indicate that the boundaries could not be deduced because of missing cDNA information. The coding sequences of transcript variant 1 and 2 of Amer2 are indicated by thick lines, non-coding regions and the splicing pattern by thin lines. The common ATG (start) and stop codons of the Amer2 isoforms are indicated. (B) Sequence comparison of human Amer1 and Amer2 transcript variant 1. Amino acid sequences were aligned using Vector NTI Advance 10 (Invitrogen). Black boxes indicate conserved APC binding sites. (C) Co-immunoprecipitation of overexpressed and endogenous APC with EGFP-tagged Amer2 after transient transfections of 293T cells as indicated. Western blottings were performed using Ali (for APC), and anti-GFP antibodies. The double band for EGFP-Amer2 might result from incomplete denaturation of the protein in the gel sample buffer.

• Supplemental Figure S3 -

  Fig. S3. Plasma membrane localization of APC-Arm and APC-Arm^N507K in the presence of Amer1 and Amer1(short). Staining of EGFP-APC-Arm, EGFP-APC-Arm^N507K (GFP fluorescence), Amer1 and Amer1(short) (anti-Flag antibody immunofluorescence) in MCF-7 cells transiently transfected as indicated.

• Supplemental Figure S4 -

  Fig. 4. Sequence comparison of the lipid binding sites of Amer1 from different species, and of Amer2. Amino acid sequences were derived from DNA sequences from the NCBI refseq database corresponding to the human Amer1 coding sequence by in silico translation and aligned using Vector NTI Advance 10 (Invitrogen). Note highly conserved basic and aromatic residues highlighted in red.

• Supplemental Figure S5 -

  Fig. 5. Efficiency of siRNAs against Amer1 and APC. (A) RT-PCR of Amer1 expression in MCF-7 cells transiently transfected with control siRNA (siGFP) or siAmer1c as indicated. RT-PCR was performed using the following sense and antisense primers for Amer1: 5′-AGGGCGGCTCAGCGGACAATAAC-3′, 5′-TCTTCAGCCTACCTGGGCCGGAT-3′ at 60°C annealing temperature, 30 cycles. GAPDH was used for normalization. Bands were quantified using the AIDA image analyzer software v. 3.52 (Raytest, Straubenhardt, Germany). (B) Western blotting for APC (antibody Ali), E-cadherin and FKBP8 from lysates of MCF-7 cells transiently transfected with siRNA oligonucleotides against either GFP (as a control), or APC. Effects of siRNAs against Amer1 on the expression of transiently expressed EGFP-tagged Amer1 (C) or EGFP-tagged rAmer1 (D) in MCF-7 cells. Note that rAmer1 is mutated in the recognition sequence of siAmer1c and therefore resistant to knockdown by siAmer1c but not siAmer1a and b.