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Figure Lengend Snippet: Pan1p binds to End3p and Sla2p. (A) Coomassie-stained gel of TAP-tagged Pan1p and its binding proteins purified in two steps using immunoglobulin G (IgG)-coupled beads and S protein beads (left panel). The TAP-tag was expressed at the Pan1p C-terminus in DDY1810 cells via a chromosomal integration (PAN1-TAP). Immunoblots showing that Sla2p is coisolated with TAP-tagged Pan1p (center and right panels). The center blot was probed with an anti-Pan1p antibody, and the right blot was probed with anti-Sla2p antibody. DDY1810 (PAN1) was used as a control. (B) Coomassie-stained gel of the purified Pan1p or Pan1p-End3p complex. TAP-tagged Pan1p alone (left lane) or TAP-tagged Pan1p and End3p (right lane) were expressed in DDY1810 and purified in two steps using IgG-coupled beads and calmodulin-coupled agarose. (C) Effect of Prk1p phosphorylation on Pan1p-End3p complex. Purified Pan1p-End3p complex was incubated with 0–0.3 μM Prk1p at 30°C, separated by SDS-PAGE, and analyzed using Coomassie blue staining. (D) Interaction between Pan1p and Sla2p in the two-hybrid analysis. 1, pDB (bait vector) + pPC86 (prey vector); 2, pDB-PAN1 and pPC86; 3, pDB and pPC86-SLA2; 4, pDB-PAN1 and pPC86-SLA2. DO LW, drop-out Leu and Trp; DO LWH, drop-out Leu, Trp, and His; 3-AT, 3-amino-1,2,4-triazole. (E) In vitro binding of Pan1p to Sla2p. Purified Pan1p was pulled down with GST-Sla2. The pelleted proteins were separated by SDS-PAGE and analyzed using Coomassie blue staining. (F) Coomassie-stained gel for a cosedimentation assay using Pan1p truncation mutants. Purified GST-Sla2p was pulled down with indicated TAP-tagged Pan1 truncation mutants and analyzed as in E.

Article Snippet: GST-Sla2 pelleted with glutathione-Sepharose beads (GE Healthcare, Waukesha, WI) was washed three times with wash buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.5% NP40) and incubated with purified Pan1p in 100 μl binding buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 5 mM EGTA, 1 mM EDTA, 7.5% glycerol, 0.5% NP40, 10 mM β-mercaptoethanol, 1 mM PMSF, and 0.5 μg/ml leupeptin, antipain, pepstatin A, and aprotinin).

Techniques: Staining, Binding Assay, Purification, Western Blot, Incubation, SDS Page, Plasmid Preparation, In Vitro