Protein Delivery into 293T using Pro-DeliverIN transfection reagent

8-10 µg protein were delivered into 293T cells using 15 µL Pro-DeliverIN transfection reagent. Image analysis were performed 14-18h after.

This article demonstrates the high efficiency of Pro-DeliverIN transfection reagent from OZ Biosciences to deliver proteins in 293T cell line.

article reference: Cell Cycle, doi:10.4161/15384101.2014.960740

Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation

Gergely Róna, Máté Borsos, Jonathan J Ellis, Ahmed M. Mehdi, Mary Christie, Zsuzsanna Környei, Máté Neubrandt, Judit Tóth, Zoltán Bozóky, László Buday, Emília Madarász, Mikael Bodén, Bostjan Kobe, Beáta G. Vértessy
Abstract
Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization segments (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle.

Pro-DeliverIN™ is an innovative reagent allowing intracellular delivery of biologically active proteins. This lipid-based formulation is the first serum compatible reagent to deliver functional proteins into living cells. Consequently, it is the only reagent which allows livering protein in vitro and in vivo.