Figure 1
From: Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein
Expression of human sialidase Neu3 in CHO-K1 cells. (a) MOCK- and NEU3-transfected cells were mechanically lysed, and the homogenates (H) were ultracentrifuged. Neu3 expression in both supernatant (S) and pellet (P) fractions was determined by Western blotting using anti-Neu3 antibody. Caveolin-1 (Cav-1) and tubulin (Tub) were used as endogenous markers of a membrane-bound and a soluble protein, respectively. (b) Fixed and permeabilized NEU3-transfected cells were immunostained with antibody against Neu3 and visualized by confocal microscopy. Cells nuclei were stained blue with Hoechst dye. (c) Ganglioside content of MOCK- and NEU3-transfected cells. Cells were incubated with antibody against GD3, antibody against GD1a or CTxβ (which binds to GM1) at 4 °C for 60 min to label the plasma membrane-associated gangliosides. Then, cells were fixed, permeabilized, incubated with antibody against Neu3 and visualized by confocal microscopy. Ganglioside content was quantified according to its fluorescence intensity using ImageJ software. Values are related to the percentage of each ganglioside in MOCK-transfected cells and presented as mean ± S.E.M. Statistical analyses were made using Student’s t test with significance being attributed at the 95% level of confidence (*P ≤ 0.05; **P ≤ 0,01; ***P ≤ 0,001). (d) Total sialidase activity in cell homogenates of MOCK- and NEU3-transfected cells towards the artificial substrate 4MU-NeuAc.
