1. Localization of the Iff8 extended protein.
Localization of the Iff8 extended protein. (A) Schema of the construction: white box, the Pga59 signal sequence fused to the V5 epitope (star); dashed box, the Iff8 conserved N-terminal domain; gray box, the Iff8 Ser/Thr-rich domain; black box, the Iff8 C terminus; white box, the Iff2 Ser/Thr-rich domain, containing the imperfect repeats of the 35-amino-acid motif TEFTTTWEVTNSDGSVSTESGIVSESGTSFTTITT shown as black circles. (B) Fixed transformed cells were directly treated with anti-V5–Cy3–coupled antibody, and immunofluorescence was observed using an Olympus BX51 microscope. (C) Western blot analysis of cell wall protein fractions from the V5 epitope-tagged native Iff8 or extended Iff8+Iff2ΔS/T recombinant strains. Lanes 1 and 3, SDS- and βME-solubilized cell wall proteins; lanes 2 and 4, β-1,6-glucanase-extracted cell wall proteins separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with monoclonal anti-V5 antibody. (D) Western blotting with monoclonal anti-V5 antibody of the released proteins either after incubation of the NaCl- and Tris-washed low-speed fraction (C1,000) in 50 mM Tris (pH 7.4) buffer alone (lane 1), in Tris buffer plus 2% βME for 1 h at 37°C (lane 2), or after incubation of the SDS-extracted cell wall fraction in H2O (lane 3) or in 30 mM NaOH for 20 h at 4°C from the V5 epitope-tagged Iff8 extended strain (lane 4).
Anita Boisramé et al. Eukaryotic Cell 2011; doi: /EC