Fig. 3.

NEU3 catalytic activity in airway ECs. A–C: A549 cells were infected with increasing multiplicities of infection (MOIs) of Ad-NEU3-HA and cultured for 48 h. A: cells were lysed and the lysates were processed for immunoblotting with anti-hemagglutinin (HA) antibody. B: cells were assayed for sialidase activity for the ganglioside mixture. C: surface biotinylated proteins were harvested and processed for HA immunoblotting. To establish that samples contained plasma membrane proteins, blots were stripped and reprobed with pan-cadherin antibody. D: A549 cells were transiently infected with Ad-NEU3 at an MOI = 100. After 48 h, the cells were transfected with NEU3-targeting or control (Ctrl) siRNAs, cultured for 24, 48, or 72 h, and lysed, and the lysates were processed for immunoblotting with anti-HA antibody. E: A549 cells were infected with Ad-NEU1-FLAG at an MOI = 100. After 48 h, the cells were transfected with either NEU1-targeting, NEU3-targeting, or control siRNAs, cultured for 48 h, and lysed, and the lysates were processed for immunoblotting with anti-FLAG antibody. In A and C, to control for endogenous NEU3 expression, the blots were stripped and reprobed with anti-NEU3 antibody. In A, D, and E, to control for protein loading and transfer, blots were stripped and reprobed for β-tubulin. Molecular mass in kDa is indicated on left. Each blot is representative of ≥3 independent experiments. F: after transfection with NEU1-targeting, NEU3-targeting, or control siRNAs, 1.0 × 10⁶ A549 cells were assayed for sialidase activity for the ganglioside mixture. Nontransfected A549 cells were simultaneously assayed as the 100% control to which other groups were compared. In B and F, vertical bars represent mean ± SE relative sialidase activity. In F, the n for each control and experimental group is indicated below each bar.