For cAMP measurements, cells grown on 12-well plates were serum-deprived for at least 3 h and stimulated with 100 nM NDP-MSH. The medium was aspirated, and the cells washed with 800 µL ice-cold PBS, lysed with 200 µL/well 0.1N HCl preheated at 70 °C, and scrapped. The mix was freeze-dried, washed with 100 µL H2O and freeze-dried again. cAMP was measured with a commercial competitive enzyme immunoassay from R&D Systems (catalog number KGE002B). Parallel dishes were used for protein determination with bicinchoninic acid. To estimate ERK activation, the levels of phosphorylated ERK (pERK) were analyzed via Western blot. Cells were solubilized in 75 µL PBS supplemented with PMSF 100 ng/mL, 1% Igepal and 1% phosphatase inhibitor mix from Calbiochem. Samples were centrifuged and a volume of supernatant containing 30 µg protein was electrophoresed and blotted as described. Blots were probed with an anti-pERK1/2 rabbit polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and stained with a chemiluminescent substrate. Comparable loading was ascertained by stripping and reprobing the membranes with anti-ERK2. Quantification of band intensity was performed with ImageJ software (available at rsb.info.nih.gov/ij ).
Kge002b
Manufactured by R&D Systems
Sourced in United States, Germany
The KGE002B is a laboratory equipment product manufactured by R&D Systems. It is used for the purpose of performing specific functions within a research or testing environment. The core function of this product is to enable certain experimental or analytical procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Cytation 5, by Agilent Technologies (2 mentions)
Mmv00, by R&D Systems (1 mentions)
Bicinchoninic acid protein assay kit, by Solarbio (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Pdonr221, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Forskolin, by MedChemExpress (1 mentions)
14 protocols using kge002b
1
cAMP and ERK Activation Quantification
2
Quantifying Intracellular cAMP Levels
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Cyclic AMP concentrations were detected by a commercial ELISA (cat# KGE002B, R&D Systems, Abingdon, UK) after lysing the cells at different time intervals (0, 5, 10, 30, and 60 min). The cell’s content of cAMP was determined following the instructions of the distributor.
3
Measuring Intracellular cAMP Levels
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Cells were incubated in medium with or without 400 μM ISO for 30 minutes and lysed in lysis buffer, a PDE inhibitor was added to the cell lysate, and the degradation of cAMP was terminated by the PDE while cells were lysed. The instantaneous cAMP concentrations in cell lysates were measured using a cAMP ELISA kit (KGE002B, R&D Systems) according to the manufacturer’s instructions.
4
Quantifying VEGF and cAMP Levels
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5
Quantifying Intracellular cAMP in BEAS-2B Cells
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The content of the intracellular cAMP in the BEAS-2B cell lysates was detected by using the cAMP enzyme immunoassay kit (KGE002B, R&D Systems, USA) according to the manufacturer’s instructions. Bicinchoninic acid Protein Assay Kit (PC0020, Solarbio, China) was used to measure the protein content of the cell lysates.
6
Generating Inducible GNAS R201C Mutant
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The GNAS active mutant was previously generated by site-directed mutagenesis of R201 to cysteine56 (link). GNASR201C cDNA was cloned into the pENTR backbone (pDONR221; ThermoFisher Scientific, 12536017) using the Gateway cloning BP reaction according to the manufacturer’s protocols (Invitrogen, 11789020). Activity of the GNASR201C active mutant was confirmed by cAMP-responsive element (CRE) luciferase assay (Dual-Glo Luciferase Assay System; Promega, E2920) or cAMP immunoassay (R&D Systems, KGE002B). The GNASR201C-pENTR vector was then recombined with the lentiviral vector pLVX-TetOne FLAG Puro (provided by the Krogan group, UCSF) using the Gateway LR reaction according to the manufacturer’s protocol (Invitrogen, 11791020). Viral particles were collected from HEK293T17 cells and concentrated by ultracentrifugation. Cells were transduced two times for 48 h and then selected with 1 μg ml–1 puromycin for 5 d. Expression of GNASR201C was induced by adding 1 μg ml–1 doxycycline.
7
Measuring Cyclic AMP Accumulation
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Cells (3x106) were plated in 60-mm dishes and cultured as described above. After 24 h, the cells were preincubated with 10 mM 3-isobutyl-1-methylxanthine (IBMX) for 1 h to prevent cAMP degradation and were then stimulated for 60 min with 100 μM formaldehyde. Cyclic AMP accumulation was quantified with the cAMP parameter assay kit (R&D systems KGE 002B) according to the manufacturer’s protocol. Cells were then frozen on dry ice and cAMP was extracted with ice-cold 65% ethanol. The extracts were dried and kept at -20°C until use. The cAMP concentration in the sample was determined by interpolation to a cAMP standard curve ranging from 0.42 to 8.57 pmol/mL.
8
Measuring cAMP in PKD Tubuloids
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PKD-/- tubuloids and EV tubuloids were cultured in organoid growth medium supplemented with 1 μM AVP. After a 3-day incubation with AVP, 5 different doses (0.1-40 μM) of tolvaptan dissolved in DMSO or DMSO (vehicle) were added in triplicates. To measure intracellular cAMP level, 72-hour treated PKD-/- tubuloids and EV tubuloids were harvested, and pelleted at 300g for 5 minutes, then incubated in 500 μl of Cell Recovery Solution on ice for 1 hour. The cells were counted in organoid growth medium via a hemocytometer and subsequently washed twice in PBS. 1 × 105 cells from each condition were transferred into 1.5-ml tubes, washed 3 times in PBS, resuspended in 125 μl of 1× Cell Lysis Buffer and frozen at-20 °C. Following two additional freeze-thaw cycles cells were centrifuged at 600g (4 °C) for 10 minutes and the supernatants were carefully transferred into 96-well plates. The cAMP levels were measured at 450 nm in a plate reader (Cytation 5, Biotek, Berlin, Germany) and assessed according to the manufacturer’s instruction of cAMP Parameter assay kit (KGE002B, R&D Systems, Wiesbaden-Nordenstadt, Germany).
9
Forskolin-mediated cAMP Quantification
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Calu‐3 cells were dissociated with 0.25% Trypsin‐EDTA, divided (2 × 106 cells/tube), and incubated with or without 10 μmol/L forskolin for 20 min. Intracellular cAMP level was estimated using a competitive immunoassay kit (Cat. No. KGE002B; R&D Systems, Minneapolis, MN) according to the manufacturer's instruction.
10
Quantifying cAMP, Estrogen, and AACT in Astrocytes
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The levels of cAMP, estrogen, and AACT were measured with ELISA kits (KGE002B, R&D Systems; ZC-32658, ZCIBIO; JL13071, Jianglaibio) following the manufacturer’s instructions. The human plasma was directly used to detect estrogen and AACT. Astrocytes were lysed in Cell Lysis Buffer by repeated freeze/thaw cycles. After brief centrifugation (600g for 10 minutes), the supernatant was used for ELISA. The levels of cAMP cAMP, estrogen, and AACT were quantified by Ascent Software for Multiskan.
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