Fig. 3

Fig. 3

The Extracellular Domains of Crb2a, Crb2b-lf, and Crb2b-sf Mediate Homophilic and Heterophilic Physical Adhesion Various combinations (indicated above the blots) of GST and His fusions of the extracellular domains of Crb2a, Crb2b-lf, and Crb2b-sf were coexpressed in sf9 cells. The cell lysates were analyzed by GST pull-down and western blotting assays. Anti-GST blots confirmed the efficient pull down of GST fusion proteins. Anti-α-tubulin blotting indicated the absence of cytoplasmic protein contamination in the pull-down fractions (Pd). The pull-down fractions were washed either with PBS (PBS) only or with PBS as well as 1 M NaCl high-salt solution (HS). (A) Pull-down analyses of pairwise and coexpressed GST- and His-fusions indicated that the extracellular domain of Crb2a, Crb2b-lf, and Crb2b-sf physically adhered to each other both homo- and heterophilically. In addition, the adhesion was strong enough to withstand high ionic washes. (B) Competition pull-down analyses revealed that Crb2b-sf^ex-GST bound to Crb2b-sf^ex-His more efficiently than it bound to Crb2b-lf^ex-His or Crb2a^ex-His, as suggested by the higher ratios between Crb2b-sf^ex-His and its Crb2b-lf^ex-His or Crb2a^ex-His competitors in the pull-down fractions than in the input fractions. (C) As specificity controls, when coexpressed with Crb2a^ex-His, Crb2b-lf^ex-His, or Crb2b-sf^ex-His, GST itself did not pull down any Crb-His fusions, indicating that these His-fusions did not bind to GST or glutathione resin nonspecifically.