Approximately 24 h following transfection, proteins were separated by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membrane (GE HealthCare, Piscataway, NJ). The sample buffer contained 0.125 M Tris?HCl, pH 6.8, 20% glycerol, and 4% SDS; the final SDS concentration was 2%. Nonspecific binding was blocked by incubation for 1 h in Tris-buffered things saline (TBS; 20 mM Tris?HCl, pH 7.5, 140 mM NaCl) that contained 5% dry milk and 0.05% Tween 20 (Bio-Rad). A previously well-characterized NBCe1-A-specific antibody (8) was used at a dilution of 1:1,000. A secondary horseradish peroxidase-conjugated species-specific antibody (JacksonImmunoresearch, West Grove, PA) was used at a dilution of 1:10,000. The bands were visualized using an ECL kit and Hyperfilm ECL (GE HealthCare).

In experiments assessing the rate of induction of NBCe1-A in response to G418, cells transiently expressing the mutant plasmid were exposed 8 h after transfection to G418 (Invitrogen, 75 ��g/ml) in DMEM/10% fetal bovine serum. The cells were incubated for various times up to 72 h at 37��C. Following G418 exposure, the cells were washed three times with ice-cold PBS and resuspended in 250 ��l lysis solution [50 mM Tris?HCl, pH 7.5, 1 ��g/ml pepstatin, and complete Mini protease inhibitor cocktail (1 tablet/2 ml, Roche, Basel, Switzerland)]. The cells were then homogenized by passing 10 times through a 25-gauge needle (BD), centrifuged at 600 g for 10 min, and then extracting protein by using 1% of n-dodecyl-��-d-maltopyranoside (DDM; Anatrace, Maumee, OH).

The samples were centrifuged at 15,000 g for 5 min at 4��C and mixed with 2 ��l of NBCe1-A-specific antibody (8) for 30 min at 4��C with gentle agitation. Protein A-Sepharose beads (GE HealthCare) preblocked with BSA (10 mg/ml) in lysis buffer containing 0.1% DDM for 1 h were equilibrated with lysis buffer containing 0.1% DDM. The samples were mixed with the protein A-Sepharose beads and incubated at 4��C for 1 h with gentle agitation. The protein was eluted with 4�� SDS sample buffer containing 400 mM DTT (final concentration 2% SDS and 100 mM DTT) at 95��C. The samples were analyzed by SDS-PAGE and immunoblotting. In the G418 removal time course protocol, the media containing G418 (75 ��g/ml) was removed 24 h after initial incubation, and the cells were assayed at various subsequent time points for NBCe1-A expression.

Quantitative PCR. Quantitative PCR was used to determine the effect of G418 on the levels of mRNA for wild-type NBCe1-A and the Q29X mutant. Total RNA was isolated from transfected HEK293-H cells using RNeasy spin columns Batimastat (RNeasy Mini Kit, Qiagen) according to the manufacturer’s instructions. The samples were treated both on-column and off-column with RNase-free DNase (Qiagen) to achieve sufficiently pure total RNA samples. First-strand cDNA was synthesized using Omniscript (Qiagen) in a 20-��l assay containing 2 ��l of 10�� RT buffer, 0.5 mM each dNTP, 1.