Fig. 2. Biochemical characterization of Ama⁺ and Ama^M109. (A) The organization of Ama with its signal sequence and three Ig domains (loops). The N terminus is towards the left. The ama^M109 and the ama^R1 mutations are also indicated. (B) Representative photomicrographs showing our S2 cell adhesion assay at t=8 hours. In the top three panels, all cells were engineered to express wild-type Nrt and were exposed to either control (left), Ama⁺-containing (center) or Ama^M109-containing (right) conditioned media. In the bottom two panels, cells were engineered to express membrane-anchored forms of Ama. (C) Quantification of our S2 cell adhesion assays. Bars represent the percent of total particles at t=0 hours counted at t=8 hours. Values represent the average of two independent experiments; error bars show the standard error of the mean. (D) Anti-Ama immunoblots. The arrow indicates the mobility of full-length Ama. Lanes 1, 2: equal amounts of Ama⁺-conditioned media or of Ama^M109-conditioned media were resolved. These lanes show that comparable amounts of Ama were present in both types of conditioned media. Lanes 3-6: immunoblots of S2 cell lysates from cell pull-down assays. Equivalent amounts of cell lysates were loaded in each lane. Lane 3: naïve S2 cells exposed to Ama⁺-conditioned media. Lane 4: Nrt-expressing S2 cells exposed to Ama⁺-conditioned media. Lane 5: naïve S2 cells exposed to Ama^M109-conditioned media. Lane 6: Nrt-expressing S2 cells exposed to Ama^M109-conditioned media. These lanes show that Ama⁺ and Ama^M109 bound specifically to S2 cells expressing Nrt. (E) Anti-Nrg immunoblots. Equivalent amounts of cell lysates were loaded in each lane. The arrow indicates the mobility of the Ama-Nrg fusion protein. Lane 1: S2 cells expressing Ama⁺-TM. Lane 2: S2 cells expressing Ama^M109-TM. These lanes show equivalent amounts of these chimeric proteins were expressed.