Figure 2.

Prostate-specific antigen (PSA) treatment enhances antibody accessibility and interleukin-2 (IL-2) functional activity in mouse IL-2/PSAcs/IL-2Rα fusion proteins. Fusion proteins (2 × and 4 × linker lengths) were treated with PSA or buffer controls for 1 hr at 37° and aliquots were analysed by immunoblot, ELISA, or by functional analysis. (a) Anti-IL-2 immunoblot analysis of fusion proteins before and after treatment with PSA. Bars indicate molecular weight markers, (+) indicates treatment with PSA, (−) indicates treatment with control buffer, and full length and predicted cleavage product containing IL-2 have been denoted (arrowheads). (b) Titration of PSA using mouse IL-2/PSAcs/2 × linker/IL-2Rα fusion protein at 37° for 1 hr and immunoblot analysis using an anti-mouse IL-2 antibody (JES6-1A12). Bars and numbers indicate molecular weight markers. Full length and cleaved fusion proteins have been denoted. The amount of PSA used in the reaction is as follows: 11·25, 5·6, 2·8, 1·4 and 0 μg. (c) PSA time–course using mouse IL-2/PSAcs/4 × linker/IL-2Rα fusion protein digested with 5 μg for 0, 1, 3, 6, 12 and 24 hr at 37° analysed by immunoblot with an anti-mouse IL-2 antibody (JES6-1A12). Medium was run as negative control (M). (d) An ELISA used to measure the amount of IL-2 before and after treatment with PSA. An apparent increase in IL-2 can be seen after PSA incubation for both fusion proteins tested. (e, f) Functional analyses of IL-2 before and after cleavage. Biologically active IL-2 from the fusion proteins was measured using the CTLL-2 functional assay. Fusion protein treated with PSA (○), or with buffer control (•), IL-2 standard (▪), and medium control (▴). The same amount of fusion protein was present in the PSA-treated and untreated samples used in the CTLL-2 assay. Points represent the average of three replicates and error bars indicate standard deviation. Representative of three independent experiments.