Journal: Nucleic Acids Research

Article Title: Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

doi: 10.1093/nar/gki169

Figure Lengend Snippet: Serum starvation differentially affects the interaction of PARN and eIF4E with the mRNA 5′ cap. ( A ) Diagrammatic representation of the luciferase mRNA and its two variants used in this study. SL–Luc had a stem loop structure inserted into its 5′ UTR. Luc–Cox contained the COX4 3′ UTR attached to the luciferase opening reading frame. All constructs were capped and contained a 40 base adenosine 3′ tail. ( B ) The RNAs outlined in panel A were labelled exclusively at their 5′ cap structure and incubated in cell extracts derived from serum-stimulated (stimulated lanes) or serum-starved cells (starved lanes). Reactions mixtures were irradiated with UV light to covalently cross-link closely associated proteins and treated with RNase. Samples were then immunoprecipitated using α-PARN (top half) or α-eIF4E (bottom half) antisera. Immunoprecipitated proteins that were cross linked to the radioactive cap were separated on polyacrylamide gels containing SDS, visualized by phosphorimaging and analysed using ImageQuant software. ( C ) Post 600 g supernatants prepared from cells grown under serum-starvation conditions (starved lanes) or in the presence of 10% serum (stimulated lanes) were incubated with m7 GTP-Sepharose beads. Beads were washed extensively and bound proteins were electrophoresed on a 10% polyacrylamide gel with SDS. Western blots were performed to assess the levels of bound PARN (top half) and eIF4E (bottom half) using specific antisera.

Article Snippet: Products were visualized by ImageQuant analysis (GE Healthcare, Amersham Biosciences).

Techniques: Luciferase, Construct, Incubation, Derivative Assay, Irradiation, Immunoprecipitation, Software, Western Blot