Figure 1. Construction and expression of rational maspin mutants.

(A) Ribbon representation of human maspin generated by UCSF Chimera (http://www.cgl.ucsf.edu/chimera/). Relative positions of reactive center loop (RCL) p′ site R³⁴⁰ and neighboring D³⁴⁶ residue are depicted as ball and stick model. (B) Schematic illustration of the wild type human maspin and four rational maspin mutants. Relative positions of RCL and D³⁴⁶ in a primary maspin sequence are depicted. The full-length, wild type maspin was denoted as maspin^WT, the truncation mutants are maspin^1–323 (C-terminal deletion starting 16 amino acids upstream of RCL), maspin^1–340 (C-terminal deletion after R³⁴⁰) and maspin^1–348 (C-terminal deletion after L³⁴⁸) and the point mutant is maspin^D346E. (C) Western blot of maspin^WT and four rational mutants re-expressed in prostate cancer cell line DU145 by adenoviral vector (multiplicity of infection, MOI = 30) in the presence or absence of proteasome inhibitor MG132 (5 µM, 6 hrs treatment). Infection of DU145 cells by adenovirus empty vector (EV) was used as a negative control. PARP was used to monitor cell viability after infection and MG132 treatment. GAPDH was used as a loading control. (D) Western blot of recombinant maspin in DU145 cells three days post-infection relative to the expression of endogenous maspin by CRL2221 cells. β tubulin was used as a loading control. (E) Growth curves of infected DU145 cells expressing either maspin^WT (•), maspin^D346E (○) or empty vector control (▪).