Expression of Sema-3A and NP-1 in tumor cells and renal cell carcinomas. (A) Western blot detection of secreted Sema-3A in concentrated (5 ×) TSNs (150 μg) from the indicated cells using anti-Sema-3A specific antibody. (B) Total lysates from cell lines indicated in panel A were immunoprecipitated with anti-Sema-3A antibody (IP Ab: Sema-3A). Immune complexes were immunoblotted (Blot Ab) with anti-Sema-3A antibody to detect Sema-3A precursor (top). The same lysates (50 μg) were also processed to detect NP-1 by immunoblotting. Expression of actin was used as internal control. Data are representative of 3 experiments. Sizes are shown in kilodaltons. (C) Total RNA (50 ng/μL) was isolated from the indicated cells and subjected to real-time PCR, as described in “Materials and methods,” to detect SEMA3A and NP1 transcripts. The mRNA levels of each gene were normalized by the mRNA levels of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Reported values are an average of 3 separate samples of each type analyzed in triplicate; bars, ± SE. (D) Immunoperoxidase staining of a paraffin-embedded tissue section of a representative human renal cell carcinoma specimen (Clear cell type, grade 3, stage I) using an anti-Sema-3A and anti-NP-1 antibody or the same dilution of a control IgG (magnification, × 40). Scarce amounts of Sema-3A and NP-1 were also detected in normal renal tissue (data not shown). Antibody localization was effected using a peroxidase reaction with 3,3-diaminobenzidine tetrahydrochloride as chromogen.