IRAS mAbs were used to detect the cellular localization of IRAS protein based on specificity characterized by western blot analysis. The pEGFPC1-IRAS plasmid was transfected into HEK293 cells. IRAS protein localization was tested by fluorescence confocal microscopy after 48 hours transfection. GFP-IRAS was primarily located in the cytoplasm in a punctate pattern (Figure 3(a) ) with concentration onto discrete loci and spot fluorescence (panel A, E, I, Journal of Biomedicine and Biotechnology M, and Q), as confirmed in previous studies [30] . The mAbs DA041, DD015, and BE073 were used as probes to detect a predominantly punctate cytosolic distribution as expected (panel B, F, G). However, the mAb BA022 homogenously detected the fluorescence distribution over the cytoplasm (panel N) and the mAb AH021 scored negatively (panel R). Merged images revealed that the GFP-IRAS fluorescence significantly coincided with the immunostaining with mAbs DA041, DD015, and BE073 (panel D, H, and L), but was not reflected by the mAbs BA022 and AH021 (panel P and T). Further, the punctate fluorescence patterns appeared to be unique to the mAbs DA041, DD015, and BE073. In addition, we also detected the endogenous IRAS protein with monoclonal antibody DA041 in PC12 cells, showing a strong signal associated with the membrane and faint signal in the cytoplasm (data not shown). These results suggest that the 3 mAbs specifically detect IRAS proteins by immunofluorescence, similar to immunoblotting results. Flow cytometry is a rapid, quantitative, multiparameter cellular analysis based on the measurement of visible and fluorescent light emission. We chose the representative mAb DA041 for further characterization based on the strong signals detected by immunoblotting and immunofluorescence. The IRAS cDNA was cloned into the mammalian expression vector PCMV-myc and was expressed in HEK293 cells. The mAb DA041 and the c-myc mAb (used as a positive control) resulted in significant increases in fluorescence intensity when compared to cells incubated with preimmune serum (Figure 3(b) ). The binding efficiencies were 30.77% by the mAb DA041 and 48.26% by the c-myc mAb. No reactivities were detectable in preimmune sera (0.01%). These results indicate mAb DA041 is effective for detecting IRAS protein by flow cytometry.