Figure 4: NGD16 promotes membrane localization of GRP78 and its binding with TIMP-1. (A, B) PC-3 and MiaPaca-2 cells were exposed to NGD16, Curcumin and vehicle at indicated concentrations for 24 h, immunostained with anti-GRP78 rabbit polyclonal antibody followed by secondary Alexa Fluor-555 (red), anti-rabbit Alexa Fluor-488 (green) and counterstained with DAPI (blue). (C) Fractionation study confirmed presence of enough GRP78 in the membrane fraction of PC-3 cells treated with NGD16 compared to untreated control. The graph was plotted taking ± SEM of results from three independent experiments and relative intensity follows the densitometry analysis of the bands. (D) Endogenous GRP78 binds to TIMP-1 in mammalian PC-3 and MiaPaca-2 cells in response to sub-toxic concentration of NGD16 for 24 h. Immunofluorescence, fractionation and IP results are representatives of atleast 3 independent experiments performed (n ≥ 3, * p< 0.05).