Isolated proteins were resolved on 10% SDS-PAGE gels, transferred to a nitrocellulose membrane by semidry transfer (Trans-Blot TurboTM Transfer System, BioRad, Hercules, United States), and analysed by immunoblotting using standard procedures, as in Novačić, et al. [45 (link)]. Blots were developed with Clarity Western ECL substrates (Biorad, Hercules, United States) and visualised with a C-DiGit Blot scanner (LI-COR Biosciences, Lincoln, United States).
For Ykl162c-a experiments, strains were grown to the exponential (OD600 2) phase in the YPD medium, and total proteins extracted as in Kushnirov [46 (link)]. Myc-tagged Ykl162c-a was probed with anti-c-Myc (1:1,000 dilution; 9E10, Santa Cruz Biotechnology, Dallas, United States) as a primary and mouse IgG κ-binding protein horseradish peroxidase (1:50,000 dilution; Santa Cruz Biotechnology, Dallas, United States) as a secondary antibody. For Hsp150-β-lactamase experiments, isolated cell wall proteins were probed with anti-HA peroxidase-conjugated antibody (1:1,250 dilution; 11,667,475,001, Roche, Basel, Switzerland).
For Ykl162c-a experiments, strains were grown to the exponential (OD600 2) phase in the YPD medium, and total proteins extracted as in Kushnirov [46 (link)]. Myc-tagged Ykl162c-a was probed with anti-c-Myc (1:1,000 dilution; 9E10, Santa Cruz Biotechnology, Dallas, United States) as a primary and mouse IgG κ-binding protein horseradish peroxidase (1:50,000 dilution; Santa Cruz Biotechnology, Dallas, United States) as a secondary antibody. For Hsp150-β-lactamase experiments, isolated cell wall proteins were probed with anti-HA peroxidase-conjugated antibody (1:1,250 dilution; 11,667,475,001, Roche, Basel, Switzerland).
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