Identification of CD9 mutant forming a weak complex with Aggrus and the tetraspanin web. (A) Schematic representation of the generated CD9 mutants: shorter extracellular loop (EC1)–deletion mutant (Δ1: 39-56 deletion); longer extracellular loop (EC2)–deletion mutants (Δ2: 114-133 deletion; Δ3: 134-153 deletion; Δ4: 154-173 deletion; and Δ5: 174-192 deletion); TM1, EC1, and TM2 deletion mutant (Δ6: 13-82 deletion); and palmitoylation-deficient point mutant (MT: C9S, C78S, C79S, C218S, and C219S). (B) CHO cells were transiently transfected with V5-tagged CD9-WT (left panels) or CD9-Δ6 (right panels) together with or without FLAG-tagged Aggrus. After solubilization with 1% CHAPS or Brij 97, the FLAG-tagged Aggrus was immunoprecipitated with an anti-FLAG agarose. The immunoprecipitants were analyzed by Western blot with an antibody to V5 tag. (C) CHO cells were transfected with plasmid encoding nothing or FLAG-Aggrus together with V5-Aggrus (left panels). In some experiments, CHO cells were transfected with plasmids encoding V5-CD9-WT or V5-CD9-Δ6 together with FLAG-CD9-WT (right panels). These cells were solubilized with lysis buffer containing 1% CHAPS. After immunoprecipitation with an anti-FLAG agarose, the immunoprecipitants were analyzed by Western blot with antibodies to V5 or FLAG. (D) CHO cells were transiently transfected with FLAG-Aggrus expressing plasmid together with plasmids encoding nothing (CHO/Aggrus), V5-CD9-WT (CHO/Aggrus+CD9-WT), or V5-CD9-Δ6 (CHO/Aggrus+CD9-Δ6). Then, cells were solubilized in lysis buffer containing 1% CHAPS or Brij 97. Each cell lysate was centrifuged in sucrose gradients and fractioned. Then, each fraction was electrophoresed and analyzed by Western blot with antibodies to Aggrus, caveolin, FLAG tag, or V5 tag. LMFs were concentrated in fraction numbers 2 through 4 (F2-F4).