. Author manuscript; available in PMC: 2008 Dec 8.

Published in final edited form as: J Cell Biochem. 2008 Oct 15;105(3):847–858. doi: 10.1002/jcb.21880

TABLE II.

Yeast Two-Hybrid Analysis of Protein–Protein Interactions

Cotransformed vector constructs		Reporter gene activation
		Ade2	His3
pGBDUC2	pACT2	ULA	ULH 0 mM	ULH 1 mM	ULH 3 mM	ULH 5 mM	ULH 10 mM
A
eEF1A1	IGFN1_Ct	10	10	10	2	1	0
eEF1A2	IGFN1_Ct	10	10	10	10	5	3
eEF1A1	MyBP-C_Ct	0	1	0	0	0	0
eEF1A2	MyBP-C_Ct	0	1	0	0	0	0
eEF1A1	IGFN1	0	5	0	0	0	0
eEF1A2	IGFN1	5	7	0	0	0	0
EF1A	IGFN1_Ct	0	2	0	0	0	0
eEF1A1-D1	IGFN1_Ct	0	0	0	0	0	0
eEF1A1-D2	IGFN1_Ct	6	10	6	0	0	0
eEF1A1-D3	IGFN1_Ct	0	0	0	0	0	0
eEF1A1-D(1 + 2)	IGFN1_Ct	10	10	10	10	10	10
eEF1A1-D(2 + 3)	IGFN1_Ct	10	7	4	0	0	0
Co-transformed vector construct		Reporter gene activation
		Ade2	His3
pGBKT7	pACT2	ULA	ULH 0 mM	ULH 1 mM	ULH 10 mM	ULH 20 mM	ULH 40 mM

B
IGFN1	Ø	10	10	9	0	0	0
IGFN1	IGFN1	10	10	10	10	10	10

Yeast strain PJ69-4A was transformed with the combination of plasmids indicated in the table and plated on SD medium lacking uracil and leucin to select for the presence of the vectors. pGBDUC2 provides a binding domain fusion protein, while pACT2 expresses an activation domain fusion protein. Four independent colonies from each transformation plate were tested for growth on ULA and ULH plates of which the latter contained different concentrations of 3-AT as indicated in mM concentrations. Growth after 6 days is expressed on a scale from 10 for maximum growth to 0 for no growth. (A) Mapping of the interaction between human eEF1A and human IGFN1 was done by testing interactions between full-length eEF1A1, eEF1A1 domains or full-length eEF1A2 and IGFN1_Ct (amino acids 183-862), full-length IGFN1 or a C-terminal fragment of myosin binding protein C slow-type (amino acids 362-1,141; MyBP-C_Ct). Controls with empty vectors were performed and showed no self-activation for any of the constructs shown in A. (B) The ability of human IGFN1 to dimerize was tested by co-transformation of pGBKT7:hIGFN1 and pACT2:hIGFN1 constructs. The pGBKT7:hIGFN1 construct provides a BD fusion protein and was preferred over pGBDUC2:hIGFN1 due to its lower level of self-activation. No self-activation was observed with the pACT2:hIGFN1 construct, which provides an AD fusion protein. The symbol Ø designates an empty vector.