To investigate the effect of altered NHE9 levels on lumenal pH in recycling endosomes, we took advantage of the excellent overlap in localization with transferrin (Figure 7B, top panel), following 60 min uptake into live cells. Fluorescence ratio imaging was done by using a combination of pH-sensitive FITC-tagged Transferrin with pH-insensitive Alexa Fluor-tagged Transferrin as control for transferrin loading, and the endosomal pH was determined from a calibration curve (Figure 6A). Relative to the control cells (pH 5.7 ± 0.22), endosomal pH in NHE9 overexpressing cells was more alkaline (pH 6.39 ± 0.054), as expected from Na+(K+)/H+ exchange mediating proton leak from the endosomes (Figure 6B). These results are consistent with increased endosomal pH observed by Nakamura et al. in COS7 cells overexpressing NHE98. Although luminal pH decreased upon knockdown of NHE9 (to pH 5.39, p=0.08; Student's t-test, n= three biological replicates; Figure 6B), the difference fell short of significance. Therefore, we examined the effect of NHE9 knockdown in primary cultured human glioma cells (Figure 6C). We did observe significant acidification of endosomes upon NHE9 knockdown (pH 6.60, p<0.05; Student's
