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. 2020 Mar 3;21(5):1737. doi: 10.3390/ijms21051737

Figure 2.

Figure 2

Expression and targeting of wild type NHE1 and mutants NHE-562, NHE-566, NHE-582, NHE-600, NHE-625, NHE-675 and NHE-700. (A) Western blot of whole cell lysates of stably transfected AP1 cells containing the indicated plasmids NHE-600, NHE-625, NHE-675 and NHE-700. Asterisk indicates a non-specific immunoreactive band that was present in all cell lysates. WT, wild type. AP1, mock transfected AP1 cells. The level of expression, relative to the wild type, is indicated below each lane. * indicates non-specific immunoreactive protein. (B) Analysis of mutant expression as in “A” with mutants NHE-562, NHE-566 and NHE-582. Exposure time was increased to allow for the visualization of the mutant expression. Arrow indicates the approximate position of the shortened NHE1 proteins. (C) Surface localization of the wild type (WT) and mutant NHE1 proteins in AP1 cells. The results are mean ± the S.E, n = at least 3 determinations. After external cell surface biotinylation, equal amounts of total cell lysates (T) and unbound intracellular lysates (I) were examined by Western blotting with anti-HA antibody to identify the NHE1 protein. WT and mutant cell lines (NHE-562, NHE-566, NHE-582, NHE-600, NHE-625, NHE-675 and NHE-700 are indicated). Lysates were from cell lines stably expressing wild type NHE1 and mutant NHE1 proteins, respectively. * indicates non-specific immunoreactive protein. (D) Summary of surface localization of WT and mutant NHE1 proteins in AP1 cells (as in “C”). * indicates values that were significantly different from the control values at p < 0.05. ^ indicates significantly values that were different from control values at p < 0.01. + indicates significantly different from NHE-566 at p < 0.05.