Interactions between sperm-associated antigen (SPAG)5 and Usher syndrome 2A isoform B (USH2AisoB). (A) Schematic protein structures of SPAG5 and the peptides encoded by deletion constructs. Numbers represent amino acids (aa; NP_006452). Protein fragments encoded by deletion constructs of SPAG5 and the intracellular domain (ICD) of USH2AisoB were used in a yeast two-hybrid assay, which identified a specific interaction between USH2AisoB (aa 5064 to 5196) and the C-terminal two coiled-coil domains (aa 973to 1193) of SPAG5. (B) Glutathione S-transferase (GST) pull-down assays showing that Flag-tagged SPAG5 is efficiently pulled down by GST-USH2AisoBICD, but not by GST alone, as detected by an anti-Flag antibody. The first lane shows 5% of the input of COS-1 cell lysate. (C) Hemagluttinin (HA)-SPAG5 aa 973-1193 from COS-1 lysates co-immunoprecipitated with GFP-fused USH2AisoBICD, but not with an unrelated HA-tagged protein (EPS8). As a positive control, HA-whirlin co-immunoprecipitated with green fluorescent protein (GFP)-fused USH2AisoBICD. The upper protein band in the middle blot represents the heavy chain of the anti-GFP antibody.