Figure 3
Validation of rabeprazole as LEM-BAF inhibitor. (a) Chemical structure of rabeprazole before and after activation at acidic pH37. R indicates the position of the disulfide bonded protein. (b) HTS assay in the presence of rabeprazole that was either not pre-activated (grey bars) or pre-activated in acidic buffer (dashed bars). (c) Inhibition of the LEM-BAF-DNA chain by rabeprazole (100 µM) under oxidizing (w/o GSH) or reducing (w/ GSH) conditions in the HTS assay. Rabeprazole sulfide was used as a control since it cannot form disulfide bridges. (d) The effect of acid activated rabeprazole on the DFR assay, which was performed with the DNA restriction enzyme HindIII. (e) ESI–MS spectra for the BAF protein after incubation with rabeprazole under reducing (w/ GSH) and oxidizing (w/o GSH) conditions. The theoretical masses of the compound-BAF adducts are indicated in the inset. (f, g) Fluorescence-activated cell sorting (FACS) analysis of HeLa cells 24 h after transfection with a GFP encoding plasmid using the transfection agent X-tremeGENE 9. The transfection was performed in the presence of rabeprazole (100 µM, with or without acid activation), using rabeprazole-free and uncomplexed DNA transfections as positive and negative controls, respectively. Results are shown as the percentage of GFP positive cells (f) and the GFP intensity normalized to the positive control (g). Statistical significance was tested with respect to the positive control. NS = not significant. B-D: Fluorescence was normalized with respect to the fluorescence intensity in absence of rabeprazole. B, C, D F, G: Mean ± SD (N = 3).
