VAT No. ESB95481909 - [email protected] | www.innoprot.com www.innoprot.com | [email protected] +34 946579925 Fax: | Tel.: +34 944005355 Bizkaia,EdifícioParque Tecnológico INNOVATIVE TECHNOLOGIESIN PITUITARY ADENYLATE CYCLASE-ACTI PITUITARY ADENYLATE

RECEPTOR INTERNALIZATIONASSAYS Product name: 502, 1ª Planta | 48160 | Derio | Bizkaia Derio | 502, 1ªPlanta|48160 BIOLOGICAL SYSTEMS, S.L.

ADCYAP1R1 Ec 50 PACAP-38: Z´: 0.73+/- 0.02 -tGFP ( VATING POLYPEPTIDETYPEIRECEPTOR

1.06 x10 PAC1 -tGFP) /U2OScellline -7 M

REF: P30214

-

VAT No. ESB95481909 [email protected] | www.innoprot.com www.innoprot.com | [email protected] +34 946579925 Fax: | Tel.: +34 944005355 Bizkaia,EdifícioParque Tecnológico INNOVATIVE TECHNOLOGIESIN validated using highlyThis reproducible assay hasbeen compounds. the by induced stimuli This cell line willallow theimage analysis ofthe following redistribution ADCYAP1R1, modulating itsactivationandthe potential agonists/antagonistsagainst Assay Cell Line has been designed toassay Innoprot’s ADCYAP1R1-tGFP Internalization terminus with tGFP . taggedintheN- Ireceptor polypeptide type human Pituitary adenylate cyclase-activating U2OScellsstablyexpressing Cell Linecontains Each vial of ADCYAP1R1 Internalization Assay agonist in a High Content Analysis (HCA).

Assay Briefly description description Briefly Assay PACAP-38 502, 1ª Planta | 48160 | Derio | Bizkaia Derio | 502, 1ªPlanta|48160 BIOLOGICAL SYSTEMS, S.L. process inside thecells. as aADCYAP1R1 Storage: HostCell: Number: DNAAccession activating polypeptide typeIreceptor FullName: Rep.Official Reference: ProductName: References:

P30214-DA P30214: Liquid Nitrogen U2OS

P30214

2vialsof3x10 ADCYAP1R1-tGFP_U2OS ADCYAP1R1-tGFP_U2OS

plexus and brain. myenteric uterus, acini, pancreatic medulla, ADCYAP1R1 isexpressedintheadrenal adenylate cyclase. coupledto itispositively and activities biological several mediating peptide (PACAP) binds pituitary adenylate cyclase activating glucagon/ family. This receptor homology with membersofthe significant membrane-associated the ADCYAP1R1 .ADCYAP1R1 isa by encoded is inhumans that protein a is PAC1 Ireceptor,alsoknown as polypeptide type Pituitary adenylate cyclase-activating : 1vialof2x10 :

About ADCYAP1R1 ADCYAP1R1 About Pituitary adenylate cyclase- Pituitary adenylate Gene BankAY366498 Gene 6 proliferativecells 6 division-arrested cells protein andshares VAT No. ESB95481909 [email protected] | www.innoprot.com www.innoprot.com | [email protected] +34 946579925 Fax: | Tel.: +34 944005355 Bizkaia,EdifícioParque Tecnológico INNOVATIVE TECHNOLOGIESIN PACAP-38. of Internalization Fig2. from BD Biosciences. Biosciences. from BD Pathwayanalyzed using855”“BDBioimager High-Content and detected were processes internalization and Activation 24h.

tested by RT-PCR (Fig.1). dilution, was andreceptorgeneexpression Resistant cloneswere Our plasmid was transfected inU2OS cells. tagged intheN-terminal with tGFPprotein. cyclase-activating polype sequence ofhumanPituitary adenylate Our expression plasmid co RT-PCR. Fig1. Activation and Internalization assay assay and Internalization Activation for ADCYAP1R1-tGFP (Ec50 =1.06 x (Ec50=1.06 for ADCYAP1R1-tGFP

Assay Characterization Characterization Assay

ADCYAP1R1

Concentrations from and 10 ADCYAP1R1 -7 502, 1ª Planta | 48160 | Derio | Bizkaia Derio | 502, 1ªPlanta|48160

M) BIOLOGICAL SYSTEMS, S.L. GAPDH obtained by limit obtainedbylimit 0 to 10 ptide type I receptor ptide typeI receptor ntaining the coding ntaining thecoding stimulated with stimulated

µ housekeeping gene housekeeping M were tested for tested M were

Z´= of internalizationvalidated with anaverage assaywas was calculated relative topositive (10 fixed andthenuclei stai were algorithms. analysis image thenucleiusing surrounding measuring theincrease fluorescence redistribution was determined Nuclei werestained with DAPIand fluorescence wasobserved aroundnucleus. 24 h. concentrations of withincreasing stimulated were protein, I receptortaggedinth type polypeptide cyclase-activating adenylate U2OS cells, stably expressing human Pituitary in ADCYAP1R1 cell line. line. cell ADCYAP1R1 in PACAP-38 response curve for Concentration Fig3. 10 dilution series (n=5). The Ec50 for PACAP-38 was˜ PACAP-38 for TheEc50 dilution (n=5). series -7 0.73+/- 0.02 f 0.02 0.73+/- M after a treatment of 24h with the agonist. Cells were were Cells theagonist. with 24h of treatment a Mafter

Assay Details Details Assay After thetreatmentanaccumulationof or High Content Screening. Screening. or High Content human PACAP-38 e N-terminuswithtGFP Cells with Cells were treated ned with DAPI. % Activity %Activity DAPI. with ned of fluorescence offluorescence µ

during M). The M). The

1.06 x 11 log 11 log