Identification of bpag1 as a tumor antigen recognized by auto-antibodies.

<p>(A) An example of the screening output. ScFv-tumor antigen complex was detected with auto-antibodies in tumor-bearing mouse serum. (B) Eight candidates were identified by MALDI-TOF mass spectrometry with statistical significance (p<0.05); expect  =  expectation value. (C) Comparison of bpag1, tbc1d13 and c7orf30 expression in NIH-3T3 cells (white bar), F10 melanoma cells (black bar) and F10 melanoma tumors (grey bar) by SYBR Green real-time PCR.</p

Overview of the rapid method for isolating auto-antibody against tumor-associated antigen (TAA) using a scFv library.

<p>The tumor-homing scFv-presenting phages were collected from tumors that were injected with a scFv library. The collected phages were infected to HB2151 for scFv secretion. The secreted scFvs from HB2151 were transferred to nitrocellulose membranes by colony lift. The membranes were incubated with tumor lysate followed by serum from a tumor-bearing mouse. The scFv-tumor protein complex was detected by auto-antibodies. The complex was digested into peptide by trypsin and analyzed using MALDI-TOF mass spectrometry for identification.</p

Expression of BPAG1 in normal human melanocytes and human melanoma cell lines.

<p>(A) The expression of BPAG1 and BPAG2 mRNA was quantified by RT-PCR in normal human melanocytes (NHM) and human melanoma cell lines A375 and G361. Normal human keratinocyte (NHK) mRNA was used as a positive control. β-actin was amplified as a loading control for cDNA. NTC; no template control. (B) The expression of BPAG1 protein was detected by IP-western blotting in human melanoma cell lines A375 and G361. A431 was used as positive control for BPAG1. The arrow indicates BPAG1.</p

Quantification of anti-BPAG1 auto-antibodies in melanoma patients.

<p>(A) The levels of anti-BPAG1 auto-antibodies in sera collected from healthy volunteers and melanoma patients were quantified using a MESACUP BP230 ELISA Kit. The INDEX values were plotted and the average INDEX values as shown (±S.E.M.) for control subjects (1.64±0.27) and melanoma patients (3.47±0.40). (B) The melanoma patients were classified using the American Joint Committee on Cancer (AJCC) 2002 staging criteria. <i>In situ</i>, stage I or stage II patients were categorized as “early”, while stage III or stage IV patients were categorized as “advanced”. The average INDEX values (±S.E.M.) of early and advanced melanoma patients were 4.14±0.83 and 3.15±0.43, respectively; the bars indicate the average INDEX value.</p

Detection and analysis of proteins that interact with KPNA2 and localization of KPNA2 in the nucleolus.

<p>Proteins that interact with KPNA2 in the cytoplasm and nucleus were purified using the TAP method and detected by silver staining. Proteins marked with arrows were analyzed by LC/MS/MS. HaCaT cells expressing GFP-TAP were used to detect nonspecific interactions. <b>a)</b> The results of LC/MS/MS were analyzed by pathway analysis using reactome (<a href="http://www.reactome.org" target="_blank">http://www.reactome.org</a>). The categories of “mRNA processing”, “ribonucleoprotein complex biogenesis”, “chromatin modification,” and “transcription” were the most significantly represented pathways. <b>b)</b> Immunohistochemistry revealed KPNA2 co-localization with UBF, a nucleolar marker.</p

Suppression of protein synthesis by combined KPNA knockdown.

<p>Under starvation conditions (0.1% fetal bovine serum), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 significantly suppressed protein synthesis after 48 h (*p<0.05) and 72 h (***p<0.01), as demonstrated by metabolic labeling with ³⁵S-methionine.</p

Suppression of ribosomal RNA synthesis by combined KPNA knockdown.

<p>Under starvation conditions (0.1% fetal bovine serum), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 significantly suppressed ribosomal RNA synthesis analyzed by reverse transcription-quantitative polymerase chain reaction (***p<0.01). The amount of pre-ribosomal RNA was reduced by about 37% after 72 h.</p

Overexpression of KPNA2 in proliferating cells.

<p>Immunohistochemistry showed KPNA2 was uniformly expressed throughout the epidermis in healthy skin, although KPNA2 overexpression was observed in the basal layer in psoriasis. In contrast, very few cells exhibited KPNA2 staining in the basal cells of atopic dermatitis. KPNA2 overexpression was observed in the tumor cells of Bowen’s disease, actinic keratosis, squamous cell carcinoma, Paget’s disease, Merkel cell carcinoma, and mycosis fungoides.</p

Lists of proteins analyzed by pathway analysis.

<p>Lists of proteins analyzed by pathway analysis.</p

Suppression of cell growth by combined KPNA knockdown.

<p>Under starvation conditions (0.1% FBS), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 suppressed cell growth after 120 h (*p<0.05). Only KPNA2 siRNA subtraction produced no change in proliferation.</p