Measuring Arf6-GTP in IQSEC2 Mutants

We measured Arf6-GTP in HEK293T cells transiently transfected with IQSEC2 carrying IQ domain mutations (A350V, A350T, A350D, R359C, or E849K IQSEC2) cloned into pCAGGS and HA-tagged Arf6 in pXS [10 (link)]. The relative amount of ARF6 bound to GTP as compared to total ARF6 was assessed using a GGA-3 pulldown assay and Western blot as previously described [46 (link)]. Bands were visualized using a LiCor Odyssey imaging system and quantified with Image Studio Lite. Each band was normalized to the untreated sham-transfected control.

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Jada R., Borisov V., Laury E., Halpert S., Levy N.S., Wagner S., Netser S., Walikonis R., Carmi I., Berlin S, & Levy A.P. (2023). Daily Brief Heat Therapy Reduces Seizures in A350V IQSEC2 Mice and Is Associated with Correction of AMPA Receptor-Mediated Synaptic Dysfunction. International Journal of Molecular Sciences, 24(4), 3924.

Publication 2023

Arf6 Assay Cells Mutations Western blot

Corresponding Organization : Technion – Israel Institute of Technology

Other organizations : University of Haifa, University of Connecticut

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Variable analysis

independent variables

IQSEC2 carrying IQ domain mutations (A350V, A350T, A350D, R359C, or E849K IQSEC2) cloned into pCAGGS

dependent variables

Relative amount of ARF6 bound to GTP as compared to total ARF6

control variables

HA-tagged Arf6 in pXS
Untreated sham-transfected control

positive controls

Not explicitly mentioned

negative controls

Untreated sham-transfected control

Annotations

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