Generation and Characterization of Novel Human IRAS Monoclonal Antibodies
Figure 3
Binding of IRAS mAbs to IRAS-expressing cells by immunofluorescence and flow cytometry analysis. (a) HEK293 cells were transfected with the plasmid pEGFPC1-IRAS. Cellular localization of IRAS was observed by scanning fluorescence confocal microscopy. Green represented EGFP fluorescence (A, E, I, M, and Q) from the GFP-IRAS fusion protein, blue (C, G, K, O, and S) fluorescence represented Hoechst stained cell nuclei, red (B, F, J, N, and R) fluorescence represented cells expressing GFP-IRAS labeled with TRITC-conjugated goat antimouse antibody using IRAS mAbs as the primary antibody, and yellow (D, H, L, P, and T) represented overlapping green and red fluorescence. All 3 panels had the same field of view. Scale bar, 100 m. (b) Samples were collected and separately analyzed by flow cytometry for the ability to bind the preimmune serum (control), the c-myc mAb, and the mAb DA041 after 48 hours transfection of PCMV-myc-IRAS. Results were expressed as histograms with the DNA content on -axis and the number of fluorescent cells on -axis. Cells were distributed in 2 populations, and the second population (R2) with high fluorescence reflected the population of transfected cells recognized by the mAb DA041.
