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. 2004 Aug;87(2):858–872. doi: 10.1529/biophysj.103.037671

FIGURE 4.

FIGURE 4

Intermolecular interaction of SKOR and GORK, and different SKOR C-terminal fragments tested in the yeast two-hybrid system. (A) Reciprocal interactions between SKOR and GORK C-termini. C-termini (SKOR-D338_T828, GORK-N324_T820) were fused to the LexA DNA binding domain of the vector pLexPD and to the GAL4 activator domain of the vector pAD-GAL4. Interactions were monitored in a drop test (10 μl, OD600 adjusted to 0.1) on Leu−-Trp−-His− medium containing 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside. Growth and blue dye formation report the physical association of the coexpressed fusion proteins. (B) Generation of fragments of the cytoplasmic SKOR C-terminus. The SKOR C-terminus was subdivided into seven regions (A–G). The position of the first amino acid of each region is indicated. Additionally, the positions D438 and E507 within region C are specified. Ten constructs were created and fused to the LexA DNA binding domain and the GAL4 activator domain, respectively. (C) Detection of protein associations in growth tests on Leu−-Trp−-His− medium, and by analyzing β-galactosidase activities. A positive answer in both tests is denoted by a “(+)”, a negative answer by a “(−)”. Combinations indicated by empty fields have not been tested. The fragments ABCDEF-, ABC----, ABCD---, ABCDE--, -BCDE--, and -BCDEFG fused to the LexA DNA binding domain showed autoactivity (not shown). Results obtained with these constructs are not presented and are not taken into account for the interpretation of the data. The presented results are representatives of three to seven independent experiments each.