Fig. 3.

Antitumor effect of P₄N-induced antiserum in immunodeficient mice and the target antigens of the antiserum. (A) Nude mice bearing CT26 cell tumors (n = 5 per group) were treated with 100 μL of PBS, PBS antisera, or P₄N antisera weekly. Tumor volumes were measured every 2 d after treatment. (B) Titers of specific anti-CT26 or JC cell antibodies for the PBS antisera or P₄N antisera on days 0, 17, 24, and 31 were measured. (C) Effects of P₄N on the changes in the isotypes of specific anti-CT26 cell antibodies were examined. The titers of these antisera (1,600× dilution) were measured for IgM, IgG1, IgG2a, IgG2b, or IgA. (D) IHC analysis of antisera bound to tumor antigens on the surface of CT26. Color development by HRP-conjugated anti-mouse antibodies indicates the antisera antibody. (E) Antisera binding to tumor antigens on the surface of CT26 cells was indirectly detected with FITC-conjugated goat anti-mouse IgG antibody. (F) Subcellular location of antigens recognized by the antisera was monitored by confocal microscopy. Alexa Fluor 568-conjugated anti-mouse Ig antibodies were used to display the presence of antisera (red). Alexa Fluor 488-Con A and DAPI were used to indicate the cell membrane (green) and the cell nucleus (blue), respectively. (G) Normal sera, PBS antisera, or P₄N anti-sera were used to probe Western blots of membrane proteins of extracted from CT26 cells. Two bands of 78 kDa and 55 kDa were visualized: E-cadherin proteins were used as a loading control. For all results, significant differences between the P₄N antisera group and the PBS antisera group were calculated and labeled with *P < 0.05. (H) Proliferation of CT26 cells treated with 1, 2, 4, or 8 μL of sera was analyzed by MTT assay. To create de–anti-CT26 antibody sera, the anti-CT26 autoantibodies from P₄N antisera were presubtracted by CT26 cell binding. The data are reported as the proliferation index. Significant differences between the P₄N-treated groups and the untreated group were indicated by *P < 0.05 (n = 5). (I) Cytotoxicity of P₄N antisera after neutralization with ATPA and GRP78 peptides. (Magnification: C and F, 400×.)