Figure 1.

Depletion of human NHE8 affects EGF degradation. (A) HeLa M-cells were transfected with either water (mock) or siRNA oligos directed against TSG101, Alix, or pools of 4 oligos against NHE6, 7, 8, or 9 at 100 nM using a double-transfection protocol. ¹²⁵I-EGF degradation over time was assayed as described in Materials and Methods. Error bars, ±SE over 10, 3, 4, 6, or 6 separate experiments for mock, NHE6-, TSG101-, NHE8-, or Alix-depleted cells, respectively; and the deviation from the mean of two separate experiments for NHE7 or 9-depleted cells. Unpaired t tests showed that mock- and NHE8-depleted cells had significant differences in EGF degradation at 60 and 120 min. *p = 0.024, **p = 0.0012. There were no significant differences between EGF degradation in the mock cells compared with cells depleted for NHE6, 7, or 9. (B) Depletion of NHE6, 7 or 9. HeLa M-cells stably expressing NHE6-HA, NHE7-HA, NHE8-HA or NHE9-myc were transfected with water (mock) or pools of siRNA oligos against NHE6, 7, 8, or 9 at 100 nM using a double-transfection protocol. Western blots were developed using either anti-HA or anti-myc antibodies (top panels). Levels of calreticulin were assessed in the same samples as loading controls (c, bottom panels).