Fig. 4

Fig. 4. Human whirlin binds directly to FRMPD4 and Kir2.3, but not to LRRC8A and LRRC8D. (A) Graphical representation of the predicted protein domain architecture of human LRRC8A, LRRC8D, whirlin, FRMPD4 and Kir2.3 (http://smart.embl-heidelberg.de). Peptides that were used in yeast two-hybrid experiments are underlined. (B) Yeast two-hybrid reporter gene activation upon binding of whirlin to candidate interaction partners. The panel shows selective yeast growth after the most stringent amino acid restriction (-WLHA). Plasmids encoding fragments of candidate interaction partners fused to a DNA binding domain (pBD) were co-expressed in yeast with a plasmid encoding full length whirlin fused to an activator domain (pAD). All combinations of peptides, except for controls, were analyzed in triplicate. (C) Analysis of β-galactosidase reporter gene activation in yeast grown on the selective medium as shown in (B). The blue colour results from the hydrolysis of the X-β-gal substrate by β-galactosidase and is indicative of an interaction between the two protein peptides. “+ control 1” and “+ control 2” are the positive controls (pAD whirlin full length with pBD usherin c-term) [11] and “- control” is the negative control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)