MRGPRX2-mediated Activation Assay

All cell culture and Lipofectamine 2000 transfection reagents were purchased from Invitrogen Life Technologies (Carlsbad, CA); recombinant human stem cell factor cytokine (hSCF) was from Peprotech (Rocky Hill, NJ); p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) was from Sigma-Aldrich (St. Louis, MO); PE conjugated anti-MRGPRX2 antibody was from Biolegend (San Diego, CA); polyclonal MRGPRX2 antibody was purchased from Novus Biologicals (Littleton, CO); PE conjugated anti-FLAG antibody, HRP labeled goat anti-rabbit IgG and β-actin antibodies were obtained from Cell Signaling Technology (Danvers, MA); SuperSignal® West PicoMaximum Sensitivity Substrate was from Thermo Scientific (Rockford, IL); Compound 48/80, and Icatibant were from AnaSpec (Fremont, CA); Nateglinide was from Tocris Biosciences and AG-30/5C was from Peptide International Inc. (Louisville, KY), respectively. MRGPRX2-Tango (Addgene #66440) and MRGPRX4-Tango (Addgene#66442) plasmids were gifts from Bryan Roth (32 (link)).

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Roy S., Ganguly A., Haque M, & Ali H. (2019). Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B2 receptor antagonist Icatibant are G protein-biased agonists for MRGPRX2 in mast cells. Journal of immunology (Baltimore, Md. : 1950), 202(4), 1229-1238.

Publication 2019

Lipofectamine 2000 transfection reagents p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) PE conjugated anti-MRGPRX2 antibody HRP labeled goat anti-rabbit IgG β-actin SuperSignal® West PicoMaximum Sensitivity Substrate Compound 48/80 Icatibant Nateglinide

Actin Ag 30 Anti antibody Anti igg Antibodies Antibody Biologicals Cell culture Compound 48 80 Cytokine Gifts Glucosamine Goat Human stem cell factor Icatibant Lipofectamine 2000 Nateglinide Novus Peptide Plasmids Rabbit Sensitivity Transfection

Corresponding Organization : University of Pennsylvania

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Variable analysis

independent variables

Cell culture and Lipofectamine 2000 transfection reagents
Recombinant human stem cell factor cytokine (hSCF)
P-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG)
Compound 48/80
Icatibant
Nateglinide
AG-30/5C
MRGPRX2-Tango and MRGPRX4-Tango plasmids

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

positive controls

PE conjugated anti-MRGPRX2 antibody
PE conjugated anti-FLAG antibody

negative controls

Not explicitly mentioned

Annotations

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