Figure 1.

Human PMN NEU Activity and NEU and PPCA Expression. (A) Increasing cell numbers of unstimulated PMNs were assayed in the presence and absence of 2DN (500 µg/ml), for NEU activity for the 4-MU-NANA substrate. Vertical bars represent mean ± SE NEU activity expressed as arbitrary fluorescence units. *, significantly increased compared with NEU activity associated with the cell-free control at p < 0.05. **, significantly decreased compared with NEU activity of 3.5 × 10⁷ PMNs in the absence of 2DN at p < 0.05. (B) Total RNA isolated from PMNs was processed for qRT-PCR for NEU1, NEU2, NEU3, NEU4, and PPCA mRNA levels. The mRNA levels for each NEU and PPCA were normalized to the 18S rRNA internal control. Each vertical bar represents mean ± SE normalized mRNA levels. (C) PMNs were lysed and the lysates, at 50 µg total cellular protein/lane, were processed for NEU1 (lanes 1–2), PPCA (lanes 3–4), NEU2 (lanes 5–6), NEU3 (lanes 7–9), and NEU4 (lanes 10–11) immunoblotting. Lysates from HL60 cells were used as a positive control for NEU1 (lane 2), whereas lysates from dHL60 cells were used as positive controls for PPCA (lane 4), NEU2 (lane 6), NEU3 (lane 8), and NEU4 (lane 11). Lysates from Ad-NEU3 infected A549 cells were also used as a NEU3 positive control (lane 9). To control for protein loading and transfer, blots were stripped and reprobed for β-actin. IB, immunoblot; IB*, immunoblot after stripping. MW in kDa indicated on left. Arrows on right indicate bands with the anticipated gel mobilities of the proteins of interest. The data generated in each panel represents experiments performed on ≥ 2 independent occasions. Cropped immunoblot images are shown.