Journal: Journal of Biological Chemistry

Article Title: The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival

doi: 10.1074/jbc.m400814200

Figure Lengend Snippet: FIG. 3. NHE1 stimulation leads to ERM activation. A, HRPT cell NHE1 was stimulated by an established NH4Cl (50 mM for 25 min at 37 °C) pulse protocol. NH4Cl causes cytosol acidification and, after placement in serum-free, NH4Cl-free medium (time 0), NHE activa- tion. Whole cell lysates were harvested at the indicated times, resolved by 4–20% SDS-PAGE to allow separation between individual ERM protein bands, and immunoblotted with anti-phospho-ERM antibodies (upper panel). The blots were stripped and reprobed with anti-ezrin IgG (lower panel). B, because NH4Cl may activate NHEs other than NHE1, HRPT cells were osmotically stimulated with isotonic medium with 100 mM sucrose, which activates NHE1 but inhibits NHE3. Whole cell lysates were then resolved by 8% SDS-PAGE and probed for the ERM- activated state by immunoblotting with anti-phospho-ERM antibodies (upper panel, doublets appear as a single large band) and for ezrin expression with anti-ezrin IgG (lower panel). C, PS120 cells, which do not express NHEs (upper panel) or PS120 transiently transfected to express wild-type NHE1 (lower panel), were preincubated with EIPA (5 M, 1 h). NHE1 was then stimulated by NH4Cl protocol. Total cell lysates were harvested at indicated times and immunoblotted for phos- pho-ERM expression. Both blots contain equal protein content/lane and were developed from the same film.

Article Snippet: Cell Lines—The HRPT human renal proximal tubule epithelial cell line (a gift from Dr. L. Racusen, Johns Hopkins University) and LLCPK1 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (Invitrogen) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin-fungizone solution (Sigma).

Techniques: Activation Assay, SDS Page, Western Blot, Expressing, Transfection

Journal: Journal of Biological Chemistry

Article Title: The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival

doi: 10.1074/jbc.m400814200

Figure Lengend Snippet: FIG. 4. NHE1 and ERM directly interact in the cytoskeleton fraction. To determine whether NHE1 and ERM physically interact, HRPT NHE1 was stimulated by NH4Cl and then permitted to recover for indicated time periods (A) or by sucrose for the indicated times (B). The cells were lysed in Nonidet P-40 buffer and centrifuged, and cy- toskeleton-rich pellets were lysed in 1% SDS buffer. The lysates were immunoprecipitated with affinity-purified anti-NHE1 IgG, resolved by 8% SDS-PAGE, and immunoblotted with anti-phospho-ERM IgG.

Article Snippet: Cell Lines—The HRPT human renal proximal tubule epithelial cell line (a gift from Dr. L. Racusen, Johns Hopkins University) and LLCPK1 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (Invitrogen) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin-fungizone solution (Sigma).

Techniques: Immunoprecipitation, Affinity Purification, SDS Page

Journal: Journal of Biological Chemistry

Article Title: The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival

doi: 10.1074/jbc.m400814200

Figure Lengend Snippet: FIG. 5. NHE1-ezrin interaction protects against apoptosis. A, to determine whether NHE1 regulates ERM proteins and PI3K, NHE1 was stimulated with sucrose (6 h at 37 °C) addition to medium in LLC-PK1 cell lines stably transfected with wild-type ezrin, constitutively active (T567D) ezrin, dominant negative ezrin (T567A), amino-terminal ezrin (N-term) ezrin, or Y353F ezrin that prevents PI3K interaction. B, wild-type, T567D, and T567A cells were incubated with or without STS (5 M for 6 h). *, p 0.05 compared with wild-type, STS-treated cells by ANOVA. C, HRPT cells were preincubated with PI3K inhibitor wortmannin (300 nM for 30 min at 37 °C) or LY294002 (20 M for 30 min at 37 °C), and then treated with STS (5 M for 6 h at 37 °C). Apoptosis was assayed by annexin V labeling in all experiments. *, p 0.05 compared with STS-treated cells by ANOVA.

Article Snippet: Cell Lines—The HRPT human renal proximal tubule epithelial cell line (a gift from Dr. L. Racusen, Johns Hopkins University) and LLCPK1 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (Invitrogen) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin-fungizone solution (Sigma).

Techniques: Stable Transfection, Transfection, Dominant Negative Mutation, Incubation, Labeling

Journal: Journal of Biological Chemistry

Article Title: The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival

doi: 10.1074/jbc.m400814200

Figure Lengend Snippet: FIG. 6. Apoptosis is regulated by Akt activity. A, HRPT cells were pretreated with Akt inhibitor (Akt I; 30 M at 37 °C) for 1 h and then incubated with STS (5 M for 6 h at 37 °C). The cells were stained with annexin V for apoptosis analysis. Similar results were observed in hypertonic sucrose-treated LLC-PK1 and HRPT cells (not shown). B, LLC-PK1 cells were transfected with 50 nM Akt siRNA or 50 nM 8 integrin siRNA (control siRNA) for 48 h. The cells were then incubated with or without STS (5 M for 6 h at 37 °C), and lysates were probed for Akt expression by immunoblot analysis. The blots were stripped and reprobed for -tubulin as a control for protein loading and nonspecific RNA interference effects. C, LLC-PK1 cells were treated as described in B, but intact cells were instead assayed for apoptosis by annexin V labeling. Similar results were observed when apoptosis was determined by DAPI staining of chromatin (not shown).

Article Snippet: Cell Lines—The HRPT human renal proximal tubule epithelial cell line (a gift from Dr. L. Racusen, Johns Hopkins University) and LLCPK1 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (Invitrogen) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin-fungizone solution (Sigma).

Techniques: Activity Assay, Incubation, Staining, Transfection, Control, Expressing, Western Blot, Labeling

Journal: Journal of Biological Chemistry

Article Title: The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival

doi: 10.1074/jbc.m400814200

Figure Lengend Snippet: FIG. 7. NHE1 stimulation regulates Akt activity. A, HRPT cell NHE1 was activated by NH4Cl pulse protocol. Whole cell lysates were harvested at indicated times and immunoblotted with anti-phospho- Akt antibodies (upper panel). The blots were stripped and reprobed with anti-Akt1 IgG (lower panel). B, HRPT cells were preincubated with EIPA (5 M, 1 h, 37 °C), and then NHE1 was activated by sucrose (100 mM for 1 h at 37 °C) addition to medium. Whole cell lysates were probed for Akt activity by immunoblotting with anti-phospho-Akt antibodies (upper panel); the blots were stripped and reprobed with anti-Akt1 antibodies (lower panel) as a loading control. The histogram depicts densitometry data from three phospho-Akt blots, expressed as the means S.E. *, p 0.05 compared with other groups by ANOVA.

Article Snippet: Cell Lines—The HRPT human renal proximal tubule epithelial cell line (a gift from Dr. L. Racusen, Johns Hopkins University) and LLCPK1 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (Invitrogen) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin-fungizone solution (Sigma).

Techniques: Activity Assay, Western Blot, Control