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. 2013 Feb 22;8(2):e57095. doi: 10.1371/journal.pone.0057095

Figure 7. Humoral response against AKAP4 in breast cancer patients.

Figure 7

A, To detect circulating anti-AKAP4 antibodies in cancer patient’s sera, purified recombinant AKAP4 protein was coated in 96-well plates and subjected to ELISA. Line X denotes the mean +2SD value of healthy female’s sera (absorbance value = 0.308) above which the patient’s sera was designated positive and below which the patient’s sera was designated as negative for anti-AKAP4 antibodies. All healthy males were found to be negative for anti-AKAP4 antibodies. B, Western blotting experiments were carried out by resolving purified AKAP4 recombinant protein on SDS-PAGE. Two representative cancer patient’s sera from each histotypes (DCIS, IDC and ILC) are shown depicting AKAP4 immunoreactivity. C, two representative samples showing AKAP4 immunoreactivity in different grades (grade 1, grade 2 and grade 3). D, two representative early stages and late stages sera samples showing reactivity against AKAP4. However, normal healthy sera failed to show AKAP4 immunoreactivity. E, Band 1, Coomassie stained recombinant purified AKAP4 protein; Band 2, recombinant AKAP4 protein probed with polyclonal anti-AKAP4 antibody (positive control); Band 3, recombinant AKAP4 protein probed with neutralized polyclonal anti-AKAP4 antibody (1∶10 diluted polyclonal anti-AKAP4 antibody pre-incubated with 15 µg/ml recombinant AKAP4 protein) resulted in loss of reactivity; Band 4, E.coli BL 21 (DE3) whole cell lysate probed with polyclonal anti-AKAP4 antibody (non-specific E.coli BL21 background) showed no immune-reactive band; Band 5 and 6, Representative neutralized sera samples of two cancer patients (positive for anti-AKAP4 antibodies) showed complete loss of reactivity. F. To demonstrate the specificity of anti-AKAP4 antibody in tissue specimens, serial sections were probed with anti-AKAP4 antibody and neutralized anti-AKAP4 antibody. Representative IDC specimen showing H&E staining (left), anti-AKAP4 antibody (Pre-neutralization; middle)) and neutralized anti-AKAP4 antibody (Post-neutralization; right).