Figure 8.

Addition of HEPES buffer partially restored G601S-hERG expression in microchannels. HEK, WT-hERG-HEK, and G601S-hERG-HEK cells were seeded at 630 cells/mm² and cultured for 48 h in microchannels. Media contained 10% FBS and 1 g/L glucose. a) Western blot analysis indicated very low expression of G601S-hERG protein in G601S-hERG-HEK cells without medium replenishment (control, lane 3). G601S-hERG expression was partially restored with frequent medium replenishment (12–24 h) (R, lane 4), and with media modified by addition of 10 mM or 25 mM HEPES buffer (lanes 5 and 6, respectively). Each lane includes samples combined from 4 channels; an equal amount of protein was loaded on to the gel in each lane as described in the methods section. b) Quantification of hERG expression from G601S-hERG-HEK cells shown in part a (lanes 4–6) normalized to expression in control (lane 3 in part a). (hERG expression was first normalized to actin and then normalized to control.) In each case the ratio is greater than 1, indicating a trend of increased hERG expression with medium replenishment and HEPES addition compared to control; * indicates that the ratio is significantly different from 1 (p<0.05). Data are the mean ± SE from 3 independent experiments.