Journal: The Journal of Biological Chemistry

Article Title: Regulation of cAMP-dependent Protein Kinases

doi: 10.1074/jbc.M110.155150

Figure Lengend Snippet: PKA RI-specific interaction of PrKX-like kinases is conserved between species. To follow PKA subunit interaction in living cells, PrKX (A), PrKY (B), Pkare (C), ΔDC2 (D), and PKAC3 (E) were each fused N-terminally with GFP2. RIα, RIIα, RIβ, and RIIβ were cloned into eukaryotic vectors fusing them C-terminally with Renilla luciferase (Rluc). For BRET experiments, COS-7 cells were co-transfected with the indicated constructs and grown for 2 days prior to the incubation with vehicle (−) or with (+) 50 μm forskolin, 500 μm IBMX dissolved in Dulbecco's PBS. BRET was measured immediately after addition of the luciferase substrate. Values are given as mean ± S.E. of at least three independent experiments, each performed with n = 6 replicates (***, p < 0.001; **, p < 0.01; *, p < 0.05). The dotted line indicates the mean background value.

Article Snippet: Two days after transfection, cells were rinsed with glucose-supplemented Dulbecco's PBS (D-PBS, Invitrogen), and treated with forskolin (50 μ m ) and 3-isobutyl-1-methylxanthine (IBMX) (500 μ m ) at final concentrations in D-PBS for 20 min. Coelenterazine 400a (Biotrend) was added immediately before BRET read-out.

Techniques: Clone Assay, Luciferase, Transfection, Construct, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of cAMP-dependent Protein Kinases

doi: 10.1074/jbc.M110.155150

Figure Lengend Snippet: Mutant PrKX (R283L) has the capacity for holoenzyme formation with RII subunits. A, this partial alignment of the αH-αI loop region of Cα and PrKX-like kinases highlights the conserved Cα residues previously found to be involved in bRIα (R352, R355) and bRIIα (R365, L366) subunit coordination as well as intramolecular stabilization of Cα (E208Cα), all shaded in black. Non-conserved and/or experimentally addressed residues in this study are shaded gray and written in black (L277Cα), red (R283PrKX, R280Pkare, R324F47F2.1b, R510DC2), and yellow (P254PKAC3, E253PKAC3). B, BRET interaction analyses of wild type and PrKXR283L with wild type or P0-site mutant R subunits were carried out as detailed in the legend to Fig. 1. Plasmids coding for GFP2-PrKX and GFP2-PrKXR283L were co-transfected with constructs expressing either RIα-Rluc or RIαA99S-Rluc (C), as in B, co-transfected with either RIβ-Rluc or RIβG99S-Rluc constructs. D, as in B, combined with either RIIα-Rluc or RIIαS99A-Rluc. E, as in B, combined with either RIIβ-Rluc or RIIβS114A-Rluc. Depicted are original BRET values (mean ± S.E.) obtained from at least three independent repeats (n = 6 wells; ***, p < 0.001; **, p < 0.01; *, p < 0.05). + indicates treatment with forskolin/IBMX, − indicates mock treatment, and the dotted line represents the mean background value.

Article Snippet: Two days after transfection, cells were rinsed with glucose-supplemented Dulbecco's PBS (D-PBS, Invitrogen), and treated with forskolin (50 μ m ) and 3-isobutyl-1-methylxanthine (IBMX) (500 μ m ) at final concentrations in D-PBS for 20 min. Coelenterazine 400a (Biotrend) was added immediately before BRET read-out.

Techniques: Mutagenesis, Transfection, Construct, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Regulation of cAMP-dependent Protein Kinases

doi: 10.1074/jbc.M110.155150

Figure Lengend Snippet: Mutation of Cα subunit influences intracellular holoenzyme dynamics. BRET analyses were carried out as detailed in the legend to Fig. 1. A, plasmids coding for GFP2-Cα and GFP2-CαL283R were co-transfected with constructs expressing either RIIα-Rluc or RIIαS99A-Rluc. B, as in A, combined with either RIIβ-Rluc or RIIβS114A-Rluc constructs. Depicted are original BRET values (mean ± S.E.) obtained from at least three independent repeats (n = 6 wells; **, p < 0.01; *, p < 0.05). +, indicates treatment with forskolin/IBMX; −, indicates mock treatment. The dotted line represents the mean background value.

Article Snippet: Two days after transfection, cells were rinsed with glucose-supplemented Dulbecco's PBS (D-PBS, Invitrogen), and treated with forskolin (50 μ m ) and 3-isobutyl-1-methylxanthine (IBMX) (500 μ m ) at final concentrations in D-PBS for 20 min. Coelenterazine 400a (Biotrend) was added immediately before BRET read-out.

Techniques: Mutagenesis, Transfection, Construct, Expressing

Journal: Acta Physiologica (Oxford, England)

Article Title: Genetic ablation of carbonic anhydrase IX disrupts gastric barrier function via claudin‐18 downregulation and acid backflux

doi: 10.1111/apha.12923

Figure Lengend Snippet: Acid secretory capacity declines in parallel with the decline in parietal cell number. Time course of acid secretory rates in isolated gastric mucosae of (a) 8‐day‐, (b) 1‐month‐ and (c) 6‐month‐old Car9 −/− and WT mice. (d) Basal and (e) maximal acid secretory rates after stimulation with forskolin ( FSK ) and IBMX were significantly lower in Car9 −/− mice than in WT up from 3 months of age. The decline in acid secretory capacity in the mucosa of Car9 −/− mice is paralleled by the decline in parietal cell numbers (see Fig. ). n = 6–7, * P < 0.05.

Article Snippet: Maximal acid secretory capacity was investigated by serosal application of 10 μ m forskolin (FSK) and 100 μ m IBMX, both purchased from Alexis Biochemicals (Lörrach, Germany).

Techniques: Isolation