Fig. 4. Interactions of KGB-1 and GLH-1. (A) Pull-down analysis. Lane 1, input kgb-1(um3) homogenate with anti-KGB-1 (C2-2) antibodies. Lane 2, input kgb-1(um3) homogenate, with anti-GLH-1. Lane 3, input N2 protein, with anti-KGB-1. Lane 4, N2 homogenate incubated with GST protein and GST beads, with anti-KGB-1. Lane 5, N2 homogenate incubated with GLH-1-GST and GST beads; reacted with anti-KGB-1. Lane 6, kgb-1(um3) homogenate incubated with GLH-1-GST and GST beads; reacted with anti-KGB-1. Lanes 4-6 are shown at a longer exposure than lanes 1-3, as the KGB-1 pulled down by GLH-1 was considerably less than the total KGB-1 in C. elegans, lane 3. (B) A schematic of 6-His-tagged GLH-1 constructs, with binding indicated (+). (C) Pull-down analysis. KGB-1-GST with GLH-1-6-His and three mutants of GLH-1, each tagged with 6-His. Lane 1, His-tagged GLH-1-K581N/L589W with KGB-1-GST. Lane 2, 6-His-tagged GLH-1-K581N/L588W with KGB-1-GST. Lane 3, 6-His-tagged GLH-1 lacking the D site (missing aa 581-588) with KGB-1-GST (on long exposures, GLH-1 is seen to weakly bind KGB-1, not shown). Lane 4, KGB-1-GST and full-length, wild-type GLH-1-6-His.