Enhancement of transfection efficiency in HEK-293 cells and MN9D cells using CombiMag.

HEK293 and MN9D cells were transfected using Lipofectamine 2000 and CombiMag, two transfection reagents associated in the Magnetofectamine Kit

This paper demonstrated the high efficiency of Magnetofectamine from OZ Biosciences to transfect cells and showed the capacity of CombiMag to enhance Lipofectamine 2000 transfection efficiency.

article reference: PLoS ONE 8(3): e59788.

Inhibition of Dopamine Transporter Activity by G Protein βγ Subunits
Jennie Garcia-Olivares, Delany Torres-Salazar,  William A. Owens, Tracy Baust, David P. Siderovski, Susan G. Amara, Jun Zhu, Lynette C. Daws, Gonzalo E. Torres

Abstract

Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.

CombiMag from OZ Biosciences is a very efficient transfection reagent based on Magnetofection™ technology.