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. 2013 Dec 5;8(12):e82395. doi: 10.1371/journal.pone.0082395

Figure 2. In vivo localization of Rbt1.

Figure 2

A/ The V5-tagged Rbt1 expressed under the control of the RBT1 promoter (VIF210) was detected by immunofluorescence after three different times of hypha induction. Fixed cells were directly treated first with anti-V5 antibodies and then with anti-mouse IgG-Cy3 coupled antibody, and immunofluorescence was observed using an Olympus BX51 microscope. B/ Western blot analysis of proteins solubilized either from the plasma membrane fraction (lanes 1 to 4) or from the cell walls (lanes 5 to 8) of yeast cells (odd numbers) or hyphae (even numbers): lanes 1, 2, 5 and 6, the strain VIF211 expressing the RBT1SL-V5 allele under the control of the ACT1 promoter (ACTIp); lanes 3 and 4 the strain VIF106 expressing DCW1-V5 under ACT1p and lanes 7 and 8, the strain VIF105 expressing IFF8-V5 under ACT1p. In lanes 1 to 4, proteins from a membrane-enriched pellet (C10000g) were solubilized in the presence of 2% SDS; in lanes 5 to 8, the cell wall fraction (C1000g) was incubated in NaAc buffer + 2U of β-1,6-glucanase for 3 hours at 37°C to solubilize GPI-anchored cell wall proteins. Samples were separated by SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with monoclonal anti-V5 antibodies.