Cellular localization of the Iff2, Iff3 (or -9), Iff5, Iff6, and Iff8 proteins. Cellular localization of the Iff2, Iff3 (or -9), Iff5, Iff6, and Iff8 proteins. (A) Western blot analysis of SDS- and βME-solubilized proteins from the cell wall fraction (C1,000) (lanes 1) and of extracted proteins after incubation of the resulting cell walls either in Na-acetate (NaAc) buffer alone (lanes 2) or in NaAc buffer plus 2 U of β-1,6-glucanase (lanes 3) from the V5 epitope-tagged Iff3, Iff5, Iff6, and Iff8 recombinant strains. Samples were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with monoclonal anti-V5 antibody. (B) Analysis of SDS- and βME-solubilized proteins from the cell wall fraction (lane 1) and of extracted proteins after incubation of the resulting cell walls either in NaAc buffer alone (lane 4), in NaAc buffer plus 2 U of β-1,6-glucanase (lanes 2 and 5), or in NaAc buffer plus 0.1 U of laminarinase (lanes 3 and 6) from the V5 epitope-tagged Iff2 strain. (Left panel) Western blotting with monoclonal anti-V5 antibody; (right panel) SDS-PAGE and EZblue staining (Sigma-Aldrich). (C) Western blot analysis of the low-speed supernatant (S1,000) (lanes 1), of the SDS-solubilized proteins from the high-speed pellet (C100,000) (lanes 2), and of the SDS- and βME-solubilized proteins from the cell wall fraction (C1,000) (lanes 3) from the GFP-tagged Sur7 strain (first panel), the V5-tagged Dcw1 strain (second panel), and the V5-Iff2 strain (third panel). Samples were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted either with monoclonal anti-GFP antibody (first panel) or monoclonal anti-V5 antibody (second and third panels). (D) Western blotting with monoclonal anti-V5 antibody of the released proteins either after incubation of the NaCl- and Tris-washed low-speed pellet (C1,000) in 50 mM Tris (pH 7.4) buffer alone (lane 1) or in Tris buffer plus 2% βME (lane 2) for 1 h at 37°C or after incubation of the SDS-extracted cell wall fraction in 30 mM NaOH for 20 h at 4°C (lane 3) or after incubation of the SDS- and βME-extracted cell wall fraction in H2O (lane 4) or in 30 mM NaOH for 20 h at 4°C (lane 5) from the V5 epitope-tagged Iff2 strain. Anita Boisramé et al. Eukaryotic Cell 2011; doi:10.1128/EC.05044-11