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Results: 4

1.
Fig 4.

Fig 4. From: CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex.

CFTR regulation by SNAP-23 requires syntaxin 1A. Whole-cell patch-clamp experiments were performed on L fibroblasts expressing CFTR plus or minus syntaxin 1A. (A) Recombinant SNAP-23 (40 μg/ml) inhibited cAMP-activated currents only in syntaxin 1A-expressing cells. Premixing GST–SNAP-23 (40 μg/ml or 0.8 μM) with GST–N-tail (20 μg/ml or 0.6 μM) partially neutralized the inhibition of currents by SNAP-23. (B) Anti-SNAP-23 IgG or Fab fragment (20 μg/ml) significantly increased cAMP-activated currents only in syntaxin 1A-expressing cells. (C and D) Representative whole-cell currents and IV curves without and with anti-SNAP-23 Fab (20 μg/ml) showing that the Fab-potentiated currents in the syntaxin 1A-expressing cells exhibit the features of CFTR-mediated currents: DIDS insensitive, DPC sensitive, and time independent. Data are expressed as mean ± SEM. Asterisk (*) indicates P < 0.05 relative to untreated control.

Estelle Cormet-Boyaka, et al. Proc Natl Acad Sci U S A. 2002 September 17;99(19):12477-12482.
2.
Fig 3.

Fig 3. From: CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex.

SNAP-23 binding to CFTR is potentiated by syntaxin 1A. (A) SNAP-23 was pulled down from Cos-7 lysates with indicated amounts of GST–N-tail and detected by immunoblotting. Negative controls were GST alone and GST-CL4 (cytosolic loop 4 of CFTR: amino acids 1034–1103). (B) Binding of SNAP-23 to the CFTR N-tail is substantially enhanced by syntaxin 1A. Shown are the results of pull-down experiments in which different concentrations of GST–N-tail were added to lysates of Cos-7 cells that were expressing SNAP-23 with or without syntaxin 1A. GST alone served as a negative control (results repeated six times). (C) Syntaxin 1A expression enhances the physical association of full-length CFTR and SNAP-23 detected by coimmunoprecipitation (IP) in mouse L fibroblasts. CFTR was immunoprecipitated with C-CFTR mAb and the immunoprecipitates were probed for SNAP-23, syntaxin 1A, and CFTR by immunoblotting (results repeated three times). The CFTR signal was overexposed.

Estelle Cormet-Boyaka, et al. Proc Natl Acad Sci U S A. 2002 September 17;99(19):12477-12482.
3.
Fig 2.

Fig 2. From: CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex.

SNAP-23 regulates CFTR-mediated chloride currents in HT29-Cl.19A epithelial cells. Whole-cell patch-clamp experiments and capacitance measurements were performed as described in Materials and Methods. (A) Inhibition of cAMP-dependent currents by GST–SNAP-23 (40 μg/ml or 0.8 μM) but not by GST–vesicle-associated membrane protein-2 (VAMP-2) (40 μg/ml or 1.1 μM). (B) Anti-SNAP-23 IgG or Fab fragment (20 μg/ml) increased cAMP-activated currents whereas preimmune IgG (20 μg/ml) had no effect. Note change in scale compared with A. Currents were recorded at +110 mV. (Inset) Current–voltage relationship for the Fab-potentiated currents in the presence and absence of 1 mM DPC. Data are expressed as mean ± SEM. Asterisk (*) indicates P < 0.05 relative to untreated control. (C) Whole-cell currents in the presence of anti-SNAP-23 IgG (20 μg/ml) showing time independence, DIDS insensitivity, and DPC sensitivity characteristic of CFTR-mediated currents. (D) Representative simultaneous recordings of cell capacitance (Cm) and membrane conductance (Gm). Each pair of traces derives from an individual HT29-Cl.19A cell. The anti-pan dynamin IgG (30) antibody and anti-SNAP-23 IgG were added at 100 and 20 μg/ml, respectively. (E) Mean changes in Cm. (F) Mean changes in Gm.

Estelle Cormet-Boyaka, et al. Proc Natl Acad Sci U S A. 2002 September 17;99(19):12477-12482.
4.
Fig 1.

Fig 1. From: CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex.

SNAP-23 is present at the apical membranes of HT29-Cl.19A colonic epithelial cells and physically interacts with CFTR. (A) Immunofluorescence localization of SNAP-23 in HT29-Cl.19A cells. Arrows indicate the apical membrane. (B) Preimmune control. (C) Coimmunoprecipitation (IP) of SNAP-23 with CFTR from HT29-Cl.19A epithelial cells using C-CFTR mAb or an equivalent amount of nonimmune IgG. Immunoprecipitates were blotted with CFTR GA1 mAb or SNAP-23 polyclonal antibody. The right lane shows a Western blot of 2% of the lysate used for the immunoprecipitations. The SNAP-23 antibody recognized only a 25- to 30-kDa band in the cell lysates characteristic of SNAP-23 protein. (D) Pull-down of CFTR with GST–SNAP-23. CFTR was expressed in Cos-7 cells (see Materials and Methods). GST–SNAP-23 or GST (100 μg) was used to pull down CFTR from 1.2 ml of Cos-7 cell lysate. Twenty microliters lysate was loaded in the right lane. The blot was probed by using GA1 antibody. Bands B and C represent immature and mature CFTR, respectively. (E) SNAP-23 binding to the amino terminal tail (N-tail) of CFTR. GST–N-tail (amino acids 1–75) or GST (each at 0.15 μM) was used to pull down MBP–SNAP-23 at the indicated concentrations. (F) Binding of MBP–SNAP-23 (0.28 μM) to GST–N-tail (0.15 μM) in the presence of anti-SNAP-23 Fab fragment or preimmune Fab (each at 0.4 μM or 10 μg/ml). All experiments shown were repeated at least three times with similar results.

Estelle Cormet-Boyaka, et al. Proc Natl Acad Sci U S A. 2002 September 17;99(19):12477-12482.

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