GABAAR Subunit Expression and Antibody Binding

Following our established protocol (Ly et al., 2018 (link)), HEK293T cells were transiently cotransfected with plasmids encoding for rat or human α1-, β3-, and γ2-subunits of GABA_AR and enhanced GFP (EGFP) or EGFP only and then stained with serial dilutions of purified mAbs. From live single cells with top 30% protein expression (evaluated by EGFP signal), the MFI of the Alexa Fluor 647–conjugated goat anti-human IgG (1:400; Life Technologies) was calculated, and nonlinear regression models were determined as above.

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Kreye J., Wright S.K., van Casteren A., Stöffler L., Machule M.L., Reincke S.M., Nikolaus M., van Hoof S., Sanchez-Sendin E., Homeyer M.A., Cordero Gómez C., Kornau H.C., Schmitz D., Kaindl A.M., Boehm-Sturm P., Mueller S., Wilson M.A., Upadhya M.A., Dhangar D.R., Greenhill S., Woodhall G., Turko P., Vida I., Garner C.C., Wickel J., Geis C., Fukata Y., Fukata M, & Prüss H. (2021). Encephalitis patient-derived monoclonal GABAA receptor antibodies cause epileptic seizures. The Journal of Experimental Medicine, 218(11), e20210012.

Publication 2021

Alexa Fluor 647–conjugated goat anti-human IgG

Alexa fluor 647 Anti igg Cells Dilutions Goat Human Mabs Plasmids Protein Subunits

Corresponding Organization :

Other organizations : German Center for Neurodegenerative Diseases, Birmingham Women’s and Children’s NHS Foundation Trust, National Health Service, Aston University, Humboldt-Universität zu Berlin, Charité - Universitätsmedizin Berlin, Freie Universität Berlin, Jena University Hospital, The Graduate University for Advanced Studies, SOKENDAI, National Institute for Physiological Sciences, National Institutes of Natural Sciences

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Variable analysis

independent variables

Plasmids encoding for rat or human α1-, β3-, and γ2-subunits of GABA_AR
EGFP or EGFP only

dependent variables

MFI of the Alexa Fluor 647–conjugated goat anti-human IgG

control variables

HEK293T cells
Serial dilutions of purified mAbs
Live single cells with top 30% protein expression (evaluated by EGFP signal)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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