Validation of the interaction between sperm-associated antigen (SPAG)5 and ninein-like protein isoform B (NINLisoB) interaction. (A) The schematic protein structure of NINLisoB and the predicted intermediate filament domain (IF; aa 656-925), encoded by a deletion construct. A liquid β-galactosidase assay identified a specific interaction between SPAG5 (aa 973-1193) and the intermediate filament (IF) domain of NINLisoB. (B) Glutathione S-transferase (GST) pull-down assay showing that Flag-tagged NINLisoB was efficiently pulled down by GST-SPAG5 (aa 973-1193), but not by GST alone, as detected by an anti-Flag antibody. The first lane shows 5% of the protein input. (C) Flag-NINLisoB co-immunoprecipitated with full-length HA-SPAG5 and the positive control HA-USH2AisoB ICD, but not with the HA-tagged unrelated protein MDA5.