Journal: PLoS ONE

Article Title: Analysis of the Physiological Activities of Scd6 through Its Interaction with Hmt1

doi: 10.1371/journal.pone.0164773

Figure Lengend Snippet: (A) Two-hybrid analysis; The yeast strain PJ69-4A was co-transformed with pGBD-c1 or pGBD-c1-SCD6 with either pGAD-c1 or pGAD-c1-HMT1 and colonies were spotted onto SC-Leu-Trp (WL) and SC-Leu-Trp-His (WLH) plates and incubated for 3 days at 30°C. (B) Co-immunoprecipitation analysis; Scd6Flag was immunoprecipitated from Hmt1myc strains harboring YCplac33-SCD6FLAG or empty vector with an anti-Flag antibody, and were probed for Scd6Flag and Hmt1myc using anti-Flag and anti-myc antibodies, respectively. Similar experiments were performed using total extracts treated with RNase A prior to immunoprecipitation. (C) In vitro binding assays; His 6 -Hmt1 was incubated with Glutathione sepharose beads that had been preloaded with equal amounts of GST or GST-Scd6. Recombinant proteins were then detected using Coomassie staining and associated His 6 -Hmt1 was detected using an anti-His antibody.

Article Snippet: Extracts were incubated with or without 200 μg/mL RNase A (Wako Pure Chemical Industries, Ltd., Japan) at room temperature for 30 min prior to immunoprecipitation.

Techniques: Transformation Assay, Incubation, Immunoprecipitation, Plasmid Preparation, In Vitro, Binding Assay, Recombinant, Staining