For all protein experiments, protein extracts were prepared as previously described (25 (link), 26 (link)). Briefly, the WT (H99) and the Ali1-GFP (CLT7) strains were incubated for 18 h at 30°C with 150 rpm shaking in YPD medium. Cells were pelleted, flash frozen on dry ice, and lysed by bead beating. The crude lysate was cleared by centrifugation at 2,500 × g at 4°C for 5 min, and the supernatant (total cell lysate) was transferred to a new tube. Total cell lysate protein concentrations were measured using a bicinchoninic acid assay (BCA).
To determine the relative abundance of Ali1 in different cellular fractions, the WT (H99) and Ali1-GFP (CLT7) strains were incubated and lysed as described above. Total cell lysates (T) were separated by ultracentrifugation at 30,000 × g for 1 h at 4°C (27 (link)). The soluble fraction (S) was transferred to a new tube, and the insoluble pellet (I) was resuspended in an equivalent volume of lysis buffer containing 1% Triton X‐100. All samples were normalized by total protein concentration. Western blots were performed as described previously using an anti‐GFP primary antibody (1/5,000 dilution; Roche), followed by an anti‐mouse peroxidase‐conjugated secondary antibody (1/25,000 dilution; Jackson Laboratory). Proteins were detected by enhanced chemiluminescence (ECL Prime Western blotting detection reagent; GE Healthcare).
To determine the relative abundance of Ali1 in different cellular fractions, the WT (H99) and Ali1-GFP (CLT7) strains were incubated and lysed as described above. Total cell lysates (T) were separated by ultracentrifugation at 30,000 × g for 1 h at 4°C (27 (link)). The soluble fraction (S) was transferred to a new tube, and the insoluble pellet (I) was resuspended in an equivalent volume of lysis buffer containing 1% Triton X‐100. All samples were normalized by total protein concentration. Western blots were performed as described previously using an anti‐GFP primary antibody (1/5,000 dilution; Roche), followed by an anti‐mouse peroxidase‐conjugated secondary antibody (1/25,000 dilution; Jackson Laboratory). Proteins were detected by enhanced chemiluminescence (ECL Prime Western blotting detection reagent; GE Healthcare).
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