Production of Antibodies against Multipass Membrane Proteins Expressed in Human Tumor Cells Using Dendritic Cell Immunization
Figure 2
hSTEAP4 immunization. (a) Flow cytometry analysis to detect anti-hSTEAP4 in sera from immunized mice. 293T cells transiently transfected with pMRX-hSTEAP4-IRES-GFP were stained with sera, followed by PE conjugated antimouse IgG. (b) Specific binding of anti-STEAP antibody in flow cytometry analysis. The upper panels show 293T cells transfected with pMRX-hSTEAP1-IRES-GFP. The lower panels show 293T cells transfected with pCAGGS-hSTEAP4-IRES-GFP. The cells were reacted with the indicated serum and stained with PE conjugated antimouse IgG. (c) Detection of hSTEAP4 expression in LNCaP cells using flow cytometry analysis. For androgen treatment, LNCaP cells were cultured with 5-DHT (M) for 24 hours. (d) hSTEAP4 expression in androgen stimulated LNCaP cells using immunofluorescent staining. Arrow indicates a stained cell. Scale bar = 20 m. (e) hSTEAP4 proteins detected by anti-hSTEAP4 monoclonal antibody (4F2; IgG2a) in 293T cells transfected with pCAGGS-hSTEAP4. Scale bar = 20 m. (f) Anti-hSTEAP4 monoclonal antibody bound to hSTEAP4 proteins on cell surface. The antibody (4F2) was added to 293T cells transfected with pCAGGS-hSTEAP4 and the cells continued to be cultured for 30 minutes. The cells were fixed and reacted with FITC conjugated antimouse IgG. Scale bar = 20 m.
