Figure 6: NHE6 relocalization to the plasma membrane is dependent on PKC.

(a) MDA-MB-231 and (b) HT-1080 cells were incubated in normoxia or hypoxia in the presence or absence of the PKC activator PDBu (100 nM). Cell lysates were analysed by western blotting using a phospho-(Ser) PKC substrate antibody. α-Tubulin was used as a loading control. The immunoblot shown is representative of three independent experiments. (c–f) HT-1080 cells were cultured for 4 h under 21% O₂ or 1% O₂ in the presence or absence of PDBu (100 nM), PKC inhibitor GF-109203x (200 nM) or vehicle (dimethylsulfoxide). Co-immunoprecipitation of endogenous (c) RACK1-PKC or (d) NHE6–RACK1 complex in HT-1080 cells. Data represent 5% of the total cell extract used for each immunoprecipitation. (e and f) Quantification of NHE6 at the (e) plasma membrane and at (f) endosomes (n=3–5 independent experiments with >80 cells per experimental condition). (g) Endosomal–lysosomal pH measurements (n=4 independent experiments with >80 cells per experimental condition). (h) Quantification of Dox within the nucleus (n=5 independent experiments with >125 cells per experimental condition). Bars represent the mean±s.e.m. (*P<0.05, **P<0.01, ***P<0.001, unpaired Student’s t-test).