Specificity of SERPINB6 mAbs in ELISA, Western blot analysis, and immunohistochemistry. (A) Sandwich ELISA with pAb anti-SERPINB6 as catching antibodies (1 μg/mL) and biotinylated mAb PI6-12 as detecting antibody. Serial dilutions of rSERPINB6, SERPINB8, or SERPINB9 were tested. Absorbance values at 450 nm (A450/540) are plotted against the concentration of recombinant serpin (pg/mL). (B) Sandwich ELISA with pAb anti-SERPINB6 as catching antibodies (1 μg/mL) and biotinylated mAb PI6-12 as detecting antibody. Serial dilutions of lysates of Jurkat cells stably transfected with cDNA encoding SERPINB6, SERPINB8, or SERPINB9 were tested. Absorbance values at 450 nm (A450/540) are plotted against the sample dilution in the system. (C) Specificity of mAb PI6-25 in Western blot analysis. Ten microliters cell lysate (4 × 105 cells) of serpin-transfected and wild-type Jurkat cells and HeLa cells was separated on 10% SDS-PAGE and subjected to Western blotting. Blots were incubated overnight with mAb PI6-25. Arrow on the right indicates 42 kDa. (D) Specificity of mAb PI6-18 in immunocytochemistry. Cytospins of Jurkat cells, either wild-type or stably transfected with cDNAs encoding SERPINB6, SERPINB8, or SERPINB9, were analyzed by immunocytochemistry using mAb PI6-18, as described in “Patients, materials, and methods.” Nuclei were counterstained with hematoxylin. Original magnification, × 630.
