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. 2009 Jan 16;284(3):1820–1830. doi: 10.1074/jbc.M804590200

FIGURE 4.

FIGURE 4.

Silencing of NHERF-2 abolishes muscarinic-mediated translocation of PMCA. NHERF-2 was silenced in HT-29 cells using siRNA plasmids Shag-FF (vector control) and Shag-274 or Shag-361, exposed to ACh (10 μm), and cell surface biotinylation was used to determine surface levels of PMCA and NHERF-2. A, representative Western blot demonstrating silencing of NHERF-2. B, Shag-274 and Shag-361 reduced endogenous NHERF-2 protein levels to 57 ± 5% and 54 ± 6% (n = 3; **, p < 0.01). C, representative Western blot showing endogenous NHERF-1 was not altered. D, representative Western blot showing changes in membrane-associated PMCA following muscarinic activation in HT-29 cells silenced for NHERF-2. E, densitometry showing changes in membrane-associated PMCA during muscarinic stimulation in NHERF-2 knock-down cells. The ACh-mediated insertion of PMCA at 60 s was abolished. Furthermore, during stimulation the levels of PMCA in silenced cells reduced at 120 s to 46 ± 5% (Shag-274) and 53 ± 7% (Shag-361) compared with control (n = 3; ***, p < 0.001). Data are expressed as mean ± S.E.