Figure 5
From: Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein
Neu3 interacts with itself via disulfide bridges and with other proteins. (a) Total homogenates of MOCK- and NEU3-transfected cells were separated by SDS-PAGE with (+) or without (−) 5% β-mercaptoethanol (βm), and Western blotted with antibody against Neu3 and antibody against endogenous TfR. (b) NEU3-transfected cells were subjected to chemical cross-linking with (+) or without (−) the membrane-impermeable reagent BS3. Then, proteins were separated by SDS-PAGE and detected by Western blotting using anti-Neu3 antibody. Cav-1 (indicated by an arrow) and GPI-YFP were used as markers of proteins not accessible and accessible to BS3, respectively. (c) NEU3-transfected cells were subjected to chemical cross-linking with (+) or without (−) the membrane-permeable reagent DSP. Then, proteins were separated by SDS-PAGE, and detected by Western blotting using anti-Neu3 antibody. Cav-1 (indicated by an arrow) was used as marker of a protein accessible to DSP. Due to the presence of a disulfide bridge in DSP molecule, the crosslinking can be reversed by the addition of βm. The molecular masses in kDa of the standard proteins are shown on the left.
