A versatile monoclonal antibody specific to human SERPINB5

Abstract

Maspin (SERPINB5) is a member of the Clade B subgroup of the large superfamily of serine protease inhibitors. It is proposed that maspin is a tumor suppressor; however, its molecular role remains to be elucidated. Here we report the characterization of a mouse monoclonal antibody directed against human maspin. This antibody, 16F7, recognizes maspin in both its native and denatured form, unlike several other commercial antibodies tested in this study. It will be a useful and versatile tool for future analyses of the biological function of maspin.

FIG. 1.

16F7 specifically detects maspin. (A) Lysate samples of COS-1 cells transiently expressing eGFP fusions of all 13 human clade B serpins or vector alone control were equally loaded onto a 12.5% SDS-PAGE and immunoblotted with 16F7. Maspin appears as a ∼60 kDa product due to eGFP tag. (B) The same blots were stripped and reprobed for GFP as a loading control. (C) Lysate samples of MCF10A cells were separated via 12.5% SDS-PAGE and immunoblotted with the indicated monoclonals or isotype control.

FIG. 2.

16F7 detects intracellular, endogenous maspin via indirect immunofluorescence. MCF10A cells were fixed with 4% paraformaldehyde and permeabilized with 10% (v/v) Triton X-100 alone (F/T; top row) or with 0.5% (v/v) SDS (middle row), or treated with 1:1 acetone-methanol (Ac/MeOH; bottom row), then probed for maspin with G167–70, 3B8.2, or 16F7. Staining by 3B8.2 is non-specific in F/T and SDS antigen retrieval methods, as shown by staining patterns in MDA-MB-231 cells, a line that does not endogenously express maspin.

FIG. 3.

16F7 immunoprecipitates native maspin. MCF10A cells were radiolabeled then lysed by either 1% NP40 (v/v) or 0.5% (v/v) SDS, and maspin immunoprecipitated from the lysates with 16F7 or other commercially available monoclonals.

Fig. 4.

16F7 detects maspin in prostate and breast tissue sections via immunohistochemistry. Maspin localizes to basal cells of normal prostate (A) and prostate intraepithelial neoplasia (PIN) (B). In normal breast (D) and ductal carcinoma in situ (DCIS) (E), maspin localizes to myoepithelial cells, and slightly in carcinoma cells. Maspin expression is decreased in grade 4–5 prostate cancer (C) and invasive breast cancer (F).