Journal: The Journal of Biological Chemistry
Article Title: PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch *
Figure Lengend Snippet: PRMT5 peptide pull-downs confirm an in vitro interaction with NHERF2. A , GST-fused PDZ domains of GRIP1 (residues 672–754), MPP7 (residues 139–220), PDZ-LIM55 (residues 2–85), NHERF1 FL (residues 1–355), NHERF2 FL (residues 1–337), SCRIB (residues 714–801), PDZ-LIM2 (residues 1–84), and GST were incubated with biotinylated PRMT5 C terminus unphosphorylated peptide. Bound proteins were detected with α-GST antibody (short and long exposure are shown). Peptide loading was assessed with HRP-conjugated streptavidin ( SA-HRP ). The Coomassie stain demonstrates roughly equal input of the GST fusion proteins. B , schematic representation of the constructs used for peptide pull-down in C. C , purified recombinant GST, GST-tagged human NHERF2 full-length (NHERF2-PDZ 1–2), PDZ1 (amino acids 1–152), PDZ2 (amino acids 107–337), and 14-3-3ϵ were incubated with biotinylated PRMT5 C terminus unphosphorylated and Thr-634-phosphorylated peptides bound to streptavidin-agarose beads and detected by α-GST. Left lane , inputs of the GST fusion proteins. D , 293T cells were transfected with constructs expressing GFP-14-3-3ϵ and Myc-PRMT5 wild type or T634A mutant. Cell lysates were then incubated with normal mouse IgG or α-Myc antibody. Immunocomplexes were captured by Protein A beads and detected by either α-Myc or α-GFP. IB , immunoblotting.
Article Snippet: 15 μg of biotin-labeled peptides were immobilized on streptavidin-agarose beads (Sigma) in peptide-binding buffer (50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1 m m EDTA, 2 m m dithiothreitol, 0.5% Nonidet P-40) overnight at 4 °C and washed three times with binding buffer to remove unbound peptides.
Techniques: In Vitro, Incubation, Staining, Construct, Purification, Recombinant, Transfection, Expressing, Mutagenesis