the presence of both NHE6 and NHE9 in cDNA extracts from astrocytes (Figure 5A) as well as in neurons (Figure 4D). Knockdown of NHE9 (by ~80%) in the astrocytes did not alter transcript levels for NHE6, although a modest compensatory increase in NHE9 levels (15%, p= 0.004; Student's t-test, n= three biological replicates) was consistently observed upon knockdown of NHE6 (Figure 5B). We also engineered lentiviral-mediated overexpression of NHE9-GFP (Figure 5B). NHE9-GFP co-localized in part with markers for the early endosome EEA1 (fractional colocalization, 0.11 ± 0.06 SD, n=46) and more extensively with the recycling endosome marker Rab11 (0.46 ± 0.25 SD, n=71) by immunofluorescence (Figure 5C–D, top and middle panel). No NHE9-GFP was observed in the late endosome, as evidenced by lack of co-localization with lysobisphosphatidic acid (−0.01 ± 0.02 SD, n=50; Figure 5C–D, bottom panel).
