Fine epitope mapping of 297-D4, 144-A8, and α-BAP31 antibodies.

<p>(A) Schematic diagram of recombinant BAP31 fragments (residues 1–207, 1–217, 1–227, 1–237, 1–245, and 1–246) used in this study. (B) Individual fusion proteins were expressed in bacteria as fusion proteins with GST tag at the N-terminus and stained with Coomassie brilliant blue after SDS-PAGE. (C-F) Western blot analysis of GST-BAP31 fusion proteins with anti-GST (C), 297-D4 (D), 144-A8 (E), and α-BAP31 (F) antibodies. The asterisks indicate partial degradation products of GST-BAP31 fusion proteins.</p

Prediction of transmembrane helices and topology of cell surface-expressed BAP31.

<p>Prediction of transmembrane helices and topology of cell surface-expressed BAP31.</p

Antibody-antibody competition binding assay.

<p>(A) hESCs were preincubated with biotinylated 297-D4 in the presence of isotype control antibody and 144-A8 prior to the addition of phycoerythrin-conjugated streptavidin. (B,C) hESCs were preincubated with α-BAP31 in the presence of isotype control antibody, 297-D4 (B), 144-A8 (C) prior to the addition of FITC-conjugated anti-rabbit IgG. Antibody binding to hESCs was then analyzed by flow cytometry. The percent of binding inhibition was calculated from mean fluorescence intensity. Statistical analysis used the Student’s t-test (*p<0.05; **p<0.001).</p

Proposed model for the membrane topology of BAP31 on the cell surface of hESCs and mESCs.

<p>Proposed model for the membrane topology of BAP31 on the cell surface of hESCs and mESCs.</p

Flow cytometric analysis of the percent expression of cell surface-expressed BAP31 on H9 hESCs.

<p>The cell surface expression of BAP31 was examined by flow cytometric analysis with various concentrations (1, 5, or 10 μg/ml) of rabbit (residues 165–246), goat (residues 125–158), or mouse (residues 137–161) anti-BAP31 antibodies. Shown are the percentages of BAP31-positive cells at the concentration of 10 μg/ml of antibodies.</p

MAb 297-D4 and MAb 144-A8 recognize BAP31 on the cell surface of hESCs.

<p>(A) Immunoprecipitation of biotinylated hESCs with α-BAP31, 297-D4, and 144-A8 antibodies. Immunoprecipitates were detected by western blots with the indicated antibodies or SA-HRP. (B) Flow cytometric analysis of hESCs with anti-TRA-1-60, α-BAP31, 297-D4, or 144-A8 antibodies.</p

BAP31 is also expressed on the surface of mESCs.

<p>(A) The epitope sequences of 297-D4 and 144-A8 are different between hESCs and mESCs. The partial C-terminal amino acid sequences were aligned between human and mouse BAP31. The epitope sequences are underlined, and the different amino acids are indicated by arrows. (B) Flow cytometry analysis of mESCs with anti-SSEA-1, 297-D4, 144-A8, and α-BAP31 antibodies. (C) Immunoprecipitation of mESCs with 297-D4, 144-A8, and α-BAP31 antibodies. Cell surface proteins of mESCs were biotinylated, immuoprecipitated, and analyzed by SA-HRP or western blot with α-BAP31 (left panels). The input samples were also analyzed by western blots (right panels). β-actin was used as a loading control.</p

Time evolutions of RMSD_init values of (A) antibody backbone C_α atoms and (B) heavy atoms of the epitope from the starting structures prepared from docking simulations.

<p>Time evolutions of RMSD_init values of (A) antibody backbone C_α atoms and (B) heavy atoms of the epitope from the starting structures prepared from docking simulations.</p

Purification of MAbs and GST-fusion proteins.

<p>(A) Purified antibodies were subjected to 12% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. (B) Western blot analysis of the purified antibodies with HRP-conjugated anti-mouse IgG antibody. (C, D) Purification of GST and GST-BAP31 fusion proteins. GST (C) and GST-BAP31 (D) fusion proteins were purified using glutathione Sepharose beads from bacterial lysates. The purified proteins were subjected to 12% SDS-PAGE gel and stained using Coomassie Brilliant Blue R-250. Arrowheads indicate GST or GST-BAP31 proteins.</p

Calculated binding mode of epitope in complementarity determining region of (B) 144-A8 and (B) 297-D4.

<p>Carbon atoms of epitope and antibody are indicated in green and cyan, respectively. Dotted lines and yellow box indicate the hydrogen bonds and the hydrophobic interactions, respectively.</p