Bap, a Biofilm Matrix Protein of Staphylococcus aureus Prevents Cellular Internalization through Binding to GP96 Host Receptor
Figure 3
Gp96 is expressed at the cell surface of MAC-T and Hep-3B cells.
A) Western-blot analysis of Gp96 expressed at the cell surface. Surface exposed proteins (SF-extract) of MAC-T, Hep-3B, Vero and GPC-16 cells were labeled with sulpho-N-hydroxysuccinimide (NHS) biotin and purified using streptavid in columns. Captured proteins were separated in 10% SDS gel, transferred onto a nitrocellulose membrane and probed with anti-Gp96. To control membrane impermeability, α-catenin was detected from the surface proteins (SF-extract) using anti-α-catenin antibodies, whose specificity was verified using total cell extracts (T-extract). B) Cellular localization of Gp96. The cellular distribution of Gp96 was analyzed by immunofluorescence using nonpermeabilized MAC-T, Hep-3B, Vero and GPC-16 cells labelled with anti-Gp96 (Gp96 ex.), phalloidin (actin) and DAPI.