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Figure 4 | Breast Cancer Research

Figure 4

From: The Na+–H+exchanger-1 induces cytoskeletal changes involving reciprocal RhoA and Rac1 signaling, resulting in motility and invasion in MDA-MB-435 cells

Figure 4

Effect of Rho family mutants on the basal and serum deprivation stimulation of Na+–H+ exchanger (NHE)1 activity. To determine the role of Rho family G-proteins in the regulation of basal and serum deprivation stimulated Na+–H+ exchanger (NHE)1 activity, monolayers were transfected with empty pBabe vector or vector containing mutated Rho family genes. After 24 hours, medium was replaced with either serum replete or deplete medium for an additional 24 hours. Intracellular pH recoveries were measured and the initial rate of the NHE1 activity calculated as Na+-dependent H+ efflux, as described in the Methods section. All measurements were conducted in nominally HCO3-free, HEPES-buffered solutions in order to measure only the NHE. (a) Effect of dominant negative (dn) Rho family mutants on the serum deprivation-dependent upregulation of NHE1 activity. (b) Effect of constitutively active (ca) RhoA and Rac1 mutants on the serum deprivation-dependent upregulation of NHE1 activity. Data for control cells are shown in the gray solid bars, whereas the stippled bars represent data for Cdc42, stripped bars represent data for Rac1 and hatched bars represent data for RhoA. Data are expressed as means ± standard error for between 9 and 20 observations in each condition. *P < 0.01, **P < 0.001 versus control values; + P < 0.01, ++ P < 0.001 versus deprived values. Insets show the level of transfection of haemagglutinin (HA)-tagged cDNA in a typical experiment by Western blot of whole cell extracts using anti-HA antibody.

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