Fig. 5

Increased intracellular sodium inhibits HDAC6 activity. a DAOY cells were incubated with increasing concentrations of ouabain for three hours and then lysed for an in vitro HDAC activity assay as described in Materials and Methods. b DAOY cells were incubated with indicated concentrations of ouabain and the protein levels of the indicated HDAC isoforms were determined by immunoblotting. Actin immunoblot ensures equal loading. c After incubation with indicated concentrations of gramicidin A, HDAC6 was immunoprecipitated with a specific antibody and then subjected to an in vitro HDAC activity assay using a fluorescent substrate. d DAOY cells were transiently transfected with Flag-HDAC6 cDNA. The cells were incubated with the indicated concentrations of gramicidin A followed by immunoprecipitation of HDAC6. The immunoprecipitates were washed extensively and then incubated with polymerized microtubules to measure deacetylase activity. The reaction was terminated by adding lysis buffer and the samples were processed for immunoblotting with indicated antibodies. e Immunoblot for HDAC6 of stable clones of DAOY cells expressing HDAC6 shRNA. Control cells were transfected with vector only and processed in parallel. A GAPDH blot ensures that equal amounts of protein were used for analysis. f The HDAC6 knockdown cells were incubated with either High Na⁺ or Low Na⁺ buffer and the trafficking patterns of EGF-positive vesicles were analyzed as described in figure legend 3. *, P < 0.0001.