Moreover, the staining pattern was similar to the wild-type transporter. Fig. 3. Immunocytochemistry. HEK293-H cells expressing NBCe1-A-Q29X (A, B, and D) or wt-NBCe1-A (C). NBCe1-A-Q29X mutant: Nomarski (A) and lack of staining with useful site NH2-terminal NBCe1-A antibody (B). C: wt-NBCe1-A is expressed on the plasma of HEK293-H cells. Treatment … Rate of induction of NBCe1-A expression induced by G418 in cells expressing NBCe1-A-Q29X. The rate of induction of NBCe1-A expression following G418 treatment is shown in Fig. 4. By 20 h following continuous exposure to G418 (75 ��g/ml), the level of protein expression was weakly detectable by immunoblot analysis. NBCe1-A was strongly expressed by 24 h and remained detectable at 72 h. Fig. 4.
Rate of induction (in hours) of NBCe1-A protein following G418 treatment of HEK293-H cells expressing the NBCe1-A-Q29X mutant. Lack of effect of G418 on NBCe1-A-Q29X message level. Although G418 is known to cause ribosomal read-through, additional experiments were done to determine whether G418 affects NBCe1-A message levels. As shown in Fig. 5, there were no significant changes in mRNA expression of either wild-type or mutant NBCe1-A in cells treated with G418. These results indicate that the expression of the full-length NBCe1-A protein in cells transfected with the NBCe1-A-Q29X mutant is not mediated by changes in mRNA levels but takes place at the level of protein translation. Fig. 5. wt-NBCe1-A and mutant mRNA expression profile in HEK293-H cells. mRNA expression was measured by real-time RT-PCR.
Data show the expression of wt-NBCe1-A and mutant NBCe1-A-Q29X mRNA relative to the expression of GAPDH mRNA in the absence and presence … Analysis of HEK293-H cell G418 content. Since the effect of G418 is mediated intracellularly, we developed an assay system to determine the content of HEK293-H cells as a function of the extracellular G418 concentration. As shown in Fig. 6, the cellular G418 content varied directly with the media G418 concentration. The mean G418 content in cells exposed to 75 ��g/ml was 15.2 �� 2.2 ng/mg protein (n = 4, cells grown in 10-cm plates). In separate experiments, G418 was removed from the media after initial induction of NBCe1-A expression (Fig. 6). Following the removal of media G418, both cellular G418 content and NBCe1-A expression by immunoblot analysis were measured as a function of time.
The results in Fig. 6 show that the total cellular content of G418 decreases slowly following its removal from the extracellular medium, suggesting that the intracellular pool of G418 is bound and/or compartmentized. The expression of NBCe1-A protein was detectable at 120 h following the removal of G418. Fig. 6. Cellular G418 content. A: HPLC assay for detecting G418 derivatized with 1-fluoro-2,4-dinitrobenzene (DNFB). The indicated concentrations of G418 were chromatographed and analyzed as described in materials and methods. Each Dacomitinib data point represents the mean … Functional studies.