List of 6 MAbs against A549 sphere cells.

<p>List of 6 MAbs against A549 sphere cells.</p

Cell surface expression of SP1-B7 antigen on various NSCLC cells.

<p>(A) Flow cytometric analysis of A549 adherent (adh A549) and sphere cells (sph A549) with SP1-B7. Red-colored population indicates secondary antibody staining as a control. (B) Flow cytometric analysis of NSCLC cell lines (NCI-H460, NCI-H1703), virus-transformed lung epithelial cell (BEAS-2B), and PBMC with SP1-B7.</p

csBAP31 is upregulated on tumorspheres.

<p>(A) Flow cytometric analysis of A549 adherent (adh A549) and sphere cells (sph A549) with SP1-B7 and α-BAP31. Red-colored population indicates secondary antibody staining as a control. (B) Immunoprecipitation of csBAP31 with SP1-B7 and α-BAP31. Cell surface proteins of A549 adherent and sphere cells were biotinylated and subjected to immunoprecipitation with isotype control antibody (Con), SP1-B7, or α-BAP31, and immunoprecipitated csBAP31 proteins were detected with SA-HRP (top and middle panels). Total BAP31 proteins were also detected in two cell lysates with α-BAP31 (lower panel). GAPDH was used as a loading control. Coomassie brilliant blue (CBB) staining of two cell lysates is also shown as a loading control (right panel).</p

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

csBAP31-positive cells show decreased survival.

<p>(A, B) csBAP31-positive and -negative A549 cells were sorted after staining with SP1-B7. (C) Live sorted cells were seeded and cultured for 9 days. Colonies were stained with crystal violet. (D) Statistical analysis of C (<i>n</i> = 10). ***, <i>p</i><0.005.</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

csBAP31 is upregulated on caspase 3/7-high A549 cells.

<p>After incubation of A549 cells without (A, B, C) or with H₂O₂ (D, E, F), A549 adherent cells were detached and incubated with SP1-B7 and PE-conjugated anti-mouse IgG. The A549 cells were then incubated with caspase 3/7 flow cytometry assay kit before analysis. To examine whether csBAP31 expression is associated with early apoptosis, caspase-low (R1) and -high cells (R2) were gated on SYTOX AADvanced-negative cells (A, D) and analyzed for the expression of csBAP31 (B, E). csBAP31 expression is presented as MFI (<i>n = 3</i>) (C, F). ***, <i>p</i><0.005.</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

csBAP31 is upregulated on annexin V-high A549 cells.

<p>A549 adherent (A, B, C) or sphere cells (D, E, F) were stained with SP1-B7, annexin V, and PI. To examine whether csBAP1 expression is associated with apoptosis, annexin V-low (R1) and -high cells (R2) were gated on PI-negative cells (A, D) and analyzed for the expression of csBAP31 (B, E). csBAP31 expression is presented as MFIs (<i>n = 3</i>) (C, F). ***, <i>p</i><0.005.</p