Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: In vivo amelioration of endogenous antitumor autoantibodies via low-dose P4N through the LTA4H/activin A/BAFF pathway
Figure Lengend Snippet: Antitumor effect of P 4 N-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 P 4 N 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 P 4 N antisera on days 0, 17, 24, and 31 were measured. ( C ) Effects of P 4 N 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 P 4 N 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 P 4 N antisera group and the PBS antisera group were calculated and labeled with * P
Article Snippet: The sections were probed with rat monoclonal anti-F4/80 (1:50 dilution; GeneTex) and anti-CD20 (1:200 dilution; Santa Cruz Biotechnology), hamster monoclonal anti-CD11c (1:25 dilution; GeneTex), rabbit monoclonal anti-CD3 (1:50 dilution; GeneTex), and rabbit polyclonal anti–TNF-α and anti–IL-8 antibodies (1:50 dilution; Assay BioTech) at 4 °C overnight, and the detection antibodies were recognized using a horseradish peroxidase (HRP)-conjugated anti-rat, anti-hamster, or anti-rabbit IgG antibody (1:1,500 dilution; Santa Cruz Biotechnology).
Techniques: Mouse Assay, Immunohistochemistry, Binding Assay, Confocal Microscopy, Western Blot, Labeling