Fig. 4. Biochemical characterization of Nrt^M100 and Nrt^M221. (A) The Nrt protein is represented. The N terminus is towards the left. The location of mutations nrt^M29, nrt^M221, nrt^M54, nrt^M2 and nrt^M100 are also shown. nrt^M54 is an 11 bp deletion that results in a frameshift (fs). (B) Graphical representation of the quantification of our S2 cell adhesion assay. 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. (C) Immunoblots of S2 cell lysates from cell pull-down assays. (Top) Anti-Nrt immunoblots. Equivalent amounts of cell lysates were loaded in each lane. The arrow shows the migration of full-length Nrt. Lane 1: naïve S2 cells. Lane 2: S2 cells engineered to express Nrt⁺. Lane 3: S2 cells engineered to express Nrt^M100. Lane 4: S2 cells engineered to express Nrt^M221. Naïve S2 cells did not express Nrt, and all Nrt proteins were expressed from the pMET plasmid at comparable levels. (Bottom) Anti-Ama immunoblots. Equivalent amounts of cell lysates were loaded in each lane. The arrow shows the migration of full-length Ama. Lane 1: naïve S2 cells. Lane 2: Nrt⁺-expressing S2 cells. Lane 3: Nrt^M100-expressing S2 cells. Lane 4: Nrt^M221-expressing S2 cells. Only S2 cells expressing wild-type Nrt bound Ama.