Chlorotoxin-Fc Fusion Inhibits Release of MMP-2 from Pancreatic Cancer Cells
Figure 3
Evaluation of M-CTX-Fc internalized by PANC-1 cells. (a) PANC-1 cells were treated with M-CTX-Fc at 37°C and 4°C and stained with FITC-labeled anti-human IgG antibody and with anti-EEA1 antibody followed by a secondary antibody against Alexa flour 555 labeled-Rabbit IgG. (b) Cell surface receptors were reversibly biotinylated with NHS-SS-Biotin and were incubated with M-CTX-Fc for 1 h. Lane 1: biotinylated cells treated with M-CTX-Fc at 4°C; Lane 2: nonbiotinylated cells treated with M-CTX-Fc at 37°C; Lane 3: biotinylated cells treated with human IgG at 37°C; Lane 4: biotinylated cells left untreated at 37°C for 1 h; Lane 5 and 6: biotinylated cells with M-CTX-Fc at 37°C in the absence and presence of 100 nM CPZ. After treatment, cells were lysed, immunopreciptated with avidin agarose, and subjected to western blot to detect M-CTX-Fc with antihuman IgG antibody. Transferrin was monitored simultaneously as the control for internalization. The intensity of M-CTX-Fc was densitometrically analyzed by ImageJ and plotted to evaluate internalization.
