Identification of septin- and Elm1-dependent localization domains in Bni5 and Western blot confirmation of Bni5 fragment interactions with four- and five-septin complexes. Related to Fig. 3. (A) Colocalization analysis of various Bni5 fragments with septins. Imaging was performed in bni5Δ, bni5Δcdc11Δ, and bni5Δelm1Δ strain backgrounds. Colocalization between GFP-tagged Bni5 fragments and Cdc3-mCherry is indicated in a table as “+” (colocalization) or “−” (no colocalization). Fragments exhibiting colocalization in at least one strain background are highlighted in green. Bold and dashed lines indicate the presence and absence of specific regions in the corresponding Bni5 variants, respectively. For overexpression of Ext, HR2, and HR3 fragments, cells were grown in methionine-depleted SC medium for 5 h; representative images of Ext-overexpressed cells were shown beside the table. Asterisk indicates Bni5 variants. Plasmids from the pUG36-Bni5* series (Table S2) were introduced into the following strains: YEF10994 (bni5∆ CDC3-mCherry), YEF12138 (bni5∆ cdc11Δ CDC3-mCherry), and YEF11277 (bni5∆ elm1∆ CDC3-mCherry). (B) Western blot analysis using an anti-Shs1 antibody confirms the presence of Shs1 in the five-septin, but not in the four-septin, complexes used in our in vitro binding experiments. (C) Western blots using an anti-GST antibody to demonstrate the ability of different Bni5 fragments to bind septin filaments in vitro. Due to inefficient transfer of large-sized proteins from polyacrylamide gel to PVDF membrane, the GST-FL (Bni5) could be observed only when more samples were loaded for the western blot analysis (left). Source data are available for this figure: SourceData FS2.