Journal: PLoS ONE

Article Title: The eEF1γ Subunit Contacts RNA Polymerase II and Binds Vimentin Promoter Region

doi: 10.1371/journal.pone.0014481

Figure Lengend Snippet: A : Yeast two hybrid assay: AH109 yeast cells were co-transformed with the indicated constructs and plated onto SD media lacking leucine and tryptophan (-LW) to verify the expression of both bait (W + ) and prey (L + ) plasmids, or onto media lacking leucine, tryptophan, histidine and adenine (-LWHA) to examine the interaction between bait and prey proteins. B : Schematic representation of full-length proteins of both bait (RPB3) and prey (eEF1γ). C : Whole cell extracts of HeLa cells transfected with either myc-eEF1γ or flag-RPB3 and myc-eEF1γ were immunoprecipitated with the anti-flag monoclonal antibody and the co-immunoprecipitation was analysed by western blot using the anti-myc monoclonal antibody. The myc-eEF1γ signal is above the heavy chain Ig band. D : Whole cell extracts of HeLa cells transfected with either myc-eEF1γ or with myc-eEF1γ and EGFP-RPB3 were immunoprecipitated with the anti-myc monoclonal antibody and the co-immunoprecipitation was analysed by western blot using the anti-EGFP monoclonal antibody. The EGFP-RPB3 signal is above the heavy chain Ig band.

Article Snippet: Full-length human Flag-RPB3 (vector-pCMV-Tag2A) (Stratagene), EGFP-RPB3 (vector-pEGFP-N) (Clontech), and RPB3 deletion mutants were generated by the use of PCR amplification and/or sub-cloning .

Techniques: Y2H Assay, Transformation Assay, Construct, Expressing, Transfection, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: The eEF1γ Subunit Contacts RNA Polymerase II and Binds Vimentin Promoter Region

doi: 10.1371/journal.pone.0014481

Figure Lengend Snippet: A : Schematic representations of the EGFP-tagged full-length RPB3 and its derived deletion mutants. The construct designation and binding activity (to eEF1γ) are indicated on the right side. B : Cell lysates from HeLa cells co-transfected with each indicated construct (EGFP, EGFP-RPB3 and EGFP-RPB3 nested deletion-mutants) and myc-eEF1γ were immunoprecipitated with the anti-myc antibody. Immunoprecipitated samples were analysed by western blot using the anti-EGFP monoclonal antibody to identify RPB3 domains involved in myc-eEF1γ interaction ( top ). The total cell lysates were immunoblotted to verify the correct expression of the transfected molecules (bottom). The asterisks mark the signal corresponding to the RPB3-a mutant (lane 3), partially covered by the heavy chain Ig band (top) and a non-specific band (bottom). C : Schematic representations of myc-tagged full-length eEF1γ and its derived deletion mutants. Constructs designation and binding activity (to RPB3) are indicated on the right side. D : Cell lysates from HeLa cells co-transfected with flag-tagged RPB3 and myc-eEF1γ deletion constructs were immunoprecipitated with the anti-flag monoclonal antibody. Co-immunoprecipitation was analysed by western blot using the anti-myc monoclonal antibody (left). The total cell lysates were immunoblotted to verify the correct expression of all the transfected molecules (right).

Article Snippet: Full-length human Flag-RPB3 (vector-pCMV-Tag2A) (Stratagene), EGFP-RPB3 (vector-pEGFP-N) (Clontech), and RPB3 deletion mutants were generated by the use of PCR amplification and/or sub-cloning .

Techniques: Derivative Assay, Construct, Binding Assay, Activity Assay, Transfection, Immunoprecipitation, Western Blot, Expressing, Mutagenesis