Fig. 4
Ubiquitination of NHE6 is increased for the ΔES mutant. a AP-1 cells transiently expressing NHE6v1HA WT or ΔES for 24 h were lysed and total levels of NHE6 were analyzed by immunoblotting with a mouse monoclonal anti-HA (α-HAm) antibody. GAPDH was measured as a loading control (panel 1, far left). The level of ubiquitinated proteins in the total cell lysates (TCL) was examined using a mouse monoclonal anti-ubiquitin (α-Ubm) antibody (panel 2). The TCL were subjected to immunoprecipitation with a non-specific rabbit polyclonal IgG (α-IgGp) antibody (panel 3) or a rabbit polyclonal anti-HA (α-HAp) antibody (panel 4) and analyzed with a mouse monoclonal α-Ubm antibody to detect the ubiquitination levels of NHE6 WT versus ΔES. The membrane from panel 4 was then stripped and reprobed with a mouse monoclonal α-HAm antibody to examine the total amount of NHE6 WT and ΔES retrieved by immunoprecipitation (panel 5). b Quantitative analysis by densitometry of the ratio of ubiquitinated to total protein abundance of NHE6v1 WT and ΔES in the immunopreciptated pellets. Values represent the mean ± S.E.M. of three separate experiments. Statistical significance was assessed using a Student’s t-test, * p < 0.01