HPSE expression and activity are heterogeneous in HMCLs. (A) Expression of HPSE in 19 HMCLs was determined using Affymetrix U133 set microarrays. □ or ■ indicates that the Affymetrix call is absent or present, respectively. (B) Correlation between Affymetrix and real-time RT-PCR HPSE expression data. For real-time RT-PCR, HPSE expression in each sample was normalized to that of GAPDH, and the XG-2 cell line was used as a reference with the arbitrary value of 100. The correlation line is indicated in the graph. (C) HMCLs were lysed, and the lysates were separated on a 12% SDS-PAGE and analyzed by Western blot with a polyclonal anti-HPSE antibody. Both the 65-kDa MW proenzyme and the 50-kDa MW active form were identified. β-Actin was used as a loading control. Results are of 1 experiment representative of 3. kDa indicates molecular weight in thousands. (D) HPSE activity was determined using an ELISA-type detection assay. HMCL lysates were incubated with b-HS and then only undegraded b-HS could bind an FGF-coated ELISA plate. Bound b-HS was detected with HRP-streptavidin followed by a colorimetric assay. HPSE activity corresponding to absorbance at 450 nm was determined by comparison with a standard curve as described in “Materials and methods.” One unit is defined as the activity that can degrade 0.063 ng b-HS when reacted at pH 5.8 at 37°C for 1 minute. The detection limit (dotted line) was 0.05 U/mL. Results are of 1 experiment representative of 3. Data are means ± SD of HPSE activity determined on triplicates.