Journal: PLoS ONE

Article Title: Multiple Orientia tsutsugamushi Ankyrin Repeat Proteins Interact with SCF1 Ubiquitin Ligase Complex and Eukaryotic Elongation Factor 1 α

doi: 10.1371/journal.pone.0105652

Figure Lengend Snippet: (A) HeLa cells transfected with Flag-Ank1U5 for 18 h were fixed, permeabilized, and stained with anti-Flag antibody together with anti-Cullin1 or anti-EF1α antibody. Merged images show the colocalization of Ank1U5 (red) with endogenous Cullin1 and EF1α (green) in the nucleus. (B) HeLa cells transfected with either vector DNA or plasmid encoding Flag-Ank1U5 were subjected to immunoblot to monitor EF1α levels 48 h after transfection. Levels of GAPDH indicate equal protein loading. (C) The effect of Ank1U5 on EF1α ubiquitination was examined by in vitro ubiquitination reaction. EF1α was immunoprecipitated from the reaction mixture in the absence or presence of the recombinant GST-Ank1U5 at the indicated amounts and subjected to immunoblotting with anti-ubiquitin antibody.

Article Snippet: Anti-EF1α, anti-Skp1, and anti-ubiquitin antibodies were obtained from Millipore (Billerica, MA).

Techniques: Transfection, Staining, Plasmid Preparation, Western Blot, Ubiquitin Proteomics, In Vitro, Immunoprecipitation, Recombinant