Table 1. -galactosidase activity of yeast two-hybrid fusion proteins with the extracellular domain of proHB-EGF.

GAL4 DNA binding domain / GAL4 activation domain / -galactosidase activity
DTREx / LTBP-3 / +
No plasmid / LTBP-3 / -
DTREx / No plasmid / -
pGBT9 / LTBP-3 / -
pVA3 / LTBP-3 / -
DTREx / LTBP-3 EGF #1-8 / +
No plasmid / LTBP-3 EGF #1-8 / -
pGBT9 / LTBP-3 EGF #1-8 / -
pVA3 / LTBP-3 EGF #1-8 / -
DTREx / Fibulin-1C / +
No plasmid / Fibulin-1C / -
pGBT9 / Fibulin-1C / -
pVA3 / Fibulin-1C / -
DTREx / Fib-1C EGF #1-5 / -
No plasmid / Fib-1C EGF #1-5 / -
pGBT9 / Fib-1C EGF #1-5 / -
pVA3 / Fib-1C EGF #1-5 / -
DTREx / Fib-1C EGF #5-COOH / +
No plasmid / Fib-1C EGF #5-COOH / -
pGBT9 / Fib-1C EGF #5-COOH / -
pVA3 / Fib-1C EGF #5-COOH / -
pGBT9 / pGAD424 / -
pVA3 / pTD1 / +

-galactosidase activity was observed as a blue color development of the yeast colonies using the filter paper assay provided by Clontech. Color development of the colonies was allowed to continue at 30C for up to 4 h. The symbols represent: +, -galactosidase activity; -, no -galactosidase activity. Blue colonies observed after 4 h were considered to be false positives. The designations are as follows: DTREx, the extracellular domain of proHB-EGF; LTBP-3, type II calcium-binding EGF-like modules # 1-8 and the carboxyl terminus of monkey LTBP-3; LTBP-3 EGF #1-8, type II calcium-binding EGF-like modules #1-8 of LTBP-3; fibulin-1C, anaphylatoxin type I repeat 2 through the carboxyl-terminus of monkey fibulin-1C; fibulin-1C EGF #1-5, type II calcium-binding EGF-like modules #1-5 of fibulin-1C; fibulin-1C EGF #5-COOH, type II calcium-binding EGF-like modules #5-9 and carboxyl-terminus of fibulin-1C. pVA3 and pTD1 are vectors provided by Clontech that contain two fusion proteins able to interact with each other and provide a positive control for the assay.

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