Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A
Fig 2
NDPK-B interaction with CFTR is regulated by cAMP.
a) cAMP-dependent NDPK-B binding to a 175-kDa protein in overlay assays. Identical immunoblots of (100 μg) probed for NDPK-B. Lanes: 1) control blot with no overlay, 2) blot overlaid with solution containing 16HBE14o- cytosol proteins (0.5 mg). 3, 4, 5) Blot overlaid with solution 2 above containing cAMP (100 μM), 8-Br-cAMP (100 μM) or N6,O2'- dioctanoyl-cAMP (Oco2cAMP) (100 μM), respectively. Cyclic AMP enhanced NDPK-B staining at 175 kDa. Results are representative of four separate experiments. b) Quantification of the band density of NDPK-B/CFTR complex at 175kDa (n = 4) * P<0.001 Student t-test. Dioctanoyl cAMP increased NDPK-B binding 20-fold. c) Recombinant NDPK-B binds to a 175-kDa protein in cAMP-dependent manner in overlay assays. Identical western blots of membrane proteins from 16HBE14o- cells (100 μg) probed for NDPK-B staining. Lanes 1) Control with no overlay. 2) Blot overlaid with solution containing 1 μg of recombinant NDPK-B. 3) Overlaid with solution 2 containing cAMP (100 μM), 4) overlaid with solution 2 containing 8-Br-cAMP (100 μM). d) Quantification of the band density of NDPK-B at 175kDa (n = 4) * P<0.001 Student t-test. Br-cAMP increased NDPK-B binding 20-fold.