To assay Paclitaxel human endothelial cells the dose dependence of intracellular G418 in HEK293-H cells, the cells were exposed to various concentrations of G418 (0, 18.75, 37.5, 75, 150, 225, 300 ��g/ml) in DMEM/10% fetal bovine serum. The cell monolayers were incubated at each G418 concentration at 37��C (5% CO2) for 24 h. After 24 h, the cells were washed three times with ice-cold PBS and resuspended in 200 ��l borate buffer (20 mM sodium borate, pH 8). The cells were then immediately homogenized using a 25-gauge needle (BD); the homogenate was centrifuged at 600 g for 10 min and then at 15,000 g for 5 min at 4��C. Cell proteins were precipitated with methanol (1:4 ratio), and the samples were incubated on ice for 1�C2 h. The samples were then centrifuged at 15,000 g for 5 min at 4��C. The supernatant from each sample was lyophilized overnight.

Standards or lyophilized samples from HEK293-H cells containing G418, in 50 ��l of borate buffer, were each mixed with 150 ��l of 0.15 M 1-fluoro-2,4-dinitrobenzene (DNFB) and incubated for 45 min at 100��C . At the end of the incubation, the liquid from the samples was completely evaporated. The samples were cooled to room temperature, dissolved in 150 ��l of acetonitrile-water (2:1, vol/vol), and injected into the Varian ProStar 210 HPLC system equipped with a ProStar 325 Dual Wavelength UV-Vis detector with the wavelengths set at 340 and 280 nm (Varian, Palo Alto, CA). Mobile phases consisted of solvent A, 0.1% TFA in water, and solvent B, 0.1% TFA in acetonitrile. Separation of the G418-DNFB conjugate was performed with a reverse-phase C18 column (Vydac 218TP54, 4.

6 �� 250 mm, Hesperia, CA), applying the linear gradient of solvent B from 0 to 100% over 100 min (flow rate: 1 ml/min). Results were expressed as a ratio of G418 to the amount of protein in the sample. Cell protein was determined using Bradford reagent (Sigma) with absorbance measured at 595 nm. In the G418 removal time course protocol, the cells were exposed to G418 (75 ��g/ml) for 24 h, after which the compound was removed from the media and intracellular G418 was assayed at various subsequent time points. Immunocytochemistry. Approximately 24 h following transfection, HEK293-H cells growing on circular coverslips were rinsed twice with 1�� PBS and processed for examination by immunofluorescence microscopy. The cells were incubated for 2 min in 1 ml of methanol (~4��C) and then rinsed twice with 1�� PBS.

A previously well-characterized NBCe1-A-specific antibody (8) was applied at 1:100 dilution in PBS for 1 h at room temperature. After several washes in PBS, goat anti-rabbit IgG conjugated with Cy3 (1:500 dilution; Jackson ImmunoResearch) was applied for 1 h at room temperature. The slides were rinsed in PBS, treated with 4% paraformaldehyde, and mounted in Crystal/Mount (Biomeda, Entinostat Foster City, CA).