Fig. 4
DP1 interacting region of DP2. a The interactions between DP1 and DP2 fragments investigated by yeast two-hybrid assay. Domain organization of DP2 is schematically shown. Cell suspensions of each strain were spotted (3 μl of 2 × 106 cell/ml) on S.D. plates without Leu, Trp, and His (middle), and Leu, Trip, His, Ade (right) for two different selection strengths. Transformants on the non-selection plate are shown at the left. (−) indicates the transformants with the bait or prey plasmid without insert DNA. The agar plates were incubated at 30 °C for 4 days, and growing cells were visualized. b DP1 and His-tagged DP2CTD were co-purified by TALON metal affinity resin. At each purification step, aliquot was fractionated and subjected to SDS-10% PAGE followed by Coomassie Brilliant Blue staining. Protein size markers were run in lane M and their sizes are indicated on the right side of the gel. TC, total cell; SS, sonication supernatant; HP, heat precipitation; HS, heat supernatant; UB, unbound; W1–8, wash fractions; E1–8, elution fractions; M, molecular weight marker. c Complex formation of DP1 and His-tagged DP2CTD analyzed by SDS-PAGE (left) and Native PAGE (right). Purified DP1 and DP1-His-tagged DP2-CTD complex (10 pmol) were subjected to SDS-10% PAGE, or Native-5% PAGE, followed by Coomassie Brilliant Blue staining. Protein size markers were run in lane M and their sizes are indicated on the left side of the gel