(A) Two 16-mer peptides (100 ng) synthesized with repeating glycine-arginine (GRG) or glycine asymmetric dimethylarginine (GmRG) were committed to SDS-PAGE electrophoresis and transferred to western blot membrane for reaction with antisera. Anti-RG antiserum was raised against the non-methylated form of the peptide and anti-mRG against the methylated version of the peptide. Pre-immune sera were taken from all animals prior to immunization with the peptides. Preimmune sera do not recognize and bind to either form of the peptide. Anti-RG (1:1000 crude serum) recognizes only the unmodified RG peptide. Anti-mRG (1:1000 crude serum) raised against the methylated form of the RG peptide (mRG), likewise, recognizes only the methylated form of the peptide. (B) Asymmetric dimethylarginine peptides, GmRG and mNucl (derived from nucleolin) and the corresponding non-methyl congeners, GRG ands Nucl, were committed to SDS-PAGE and western blotting with anti-mRG (IgG-purified; 1:2000). (C) GRG peptide is recognized by anti-mRG only after incubation with recombinant PRMT1 (1 μg) and SAM (100 μM). (D) The mitochondrial RNA binding protein from T. brucei (RBP16) was expressed as a fusion with maltose binding protein (MBP-RBP16). MBP-RBP16 (1 μg) was incubated in Tris buffer (pH 8.0) containing 100 μM SAM and either, no further additions (---), 10 μg of a PC12 cell nuclear extract (PC12), 100 μg of a whole cell extract of T. brucei (WC) or 50 μg of a T. brucei mitochondrial extract (mito). The reaction tube contents were separated on a 10% SDS-PAGE gel and transferred to PVDF membrane. The membrane was probed with anti-mRG. A barely detectable signal is present in the lane containing RBP plus SAM only. The lane containing PC12 cell extract exhibits a 2.5-fold greater binding of anti-mRG. When T. brucei extracts are present in the incubation mixture, the reactivity of anti-GmRG increased 10-fold (WC) and 18-fold (mito).

Anti-mRG (1:2000) and anti-nucleolin (1:500) antibodies were incubated with methanol-fixed PC12 cells. Fluorescently tagged secondary antibodies against rabbit and mouse IgG were used to detect the locations of asymmetric dimethylarginine and nucleolin, respectively. Panel A, anti-mRG; panel B, anti-nucleolin; panel C, the two images merged. Confocal images were obtained through a 63x oil immersion objective. The white scale bar in panel C is 10 micrometers.

(A) Equal numbers of T. brucei cells of the procyclic (PF) and the bloodstream (BF) forms of the parasite were collected and duplicate aliquots committed to western blot membranes for reaction with anti-mRG (1:5000) and anti-RG (1:5000). The GmRG and GRG peptides (20 ng) were included as positive and negative controls. The electrophoretic migration of molecular mass markers is indicated in kilodaltons between the two membrane images. (B) PC12 cells were cultured without NGF (--) or with NGF for three days (3d). High salt extracts of isolated PC12 cell nuclei were prepared as described in Materials and Methods and run on SDS-PAGE gels for western blot analysis. Duplicate aliquots of the same protein extracts from −NGF or +NGF samples were loaded in each lane of a 10% SDS-PAGE gel. Separated proteins were transferred to a PVDF membrane and probed with anti-RG (1:1000) and anti-mRG (1:1000).

NGF was added to PC12 cell cultures for five minutes (5m), eighteen hours (18h), twenty days (20d) or cells were cultured without added NGF (−). Total cell lysates were prepared and the protein loads that were committed to immunoprecipitation from each condition were equalized based on the Biorad protein dye reagent assay. Anti-mRG or IgG from normal rabbit serum (NRS) was added to the samples as indicated. The immunoprecipitated material was collected in SDS sample buffer and run on a 10% SDS-PAGE gel followed by western blot with anti-hnRNPA1. A portion of the equalized starting cell lysates (10%; input) was blotted in lanes 7 and 8 to confirm the presence of equal amounts of hnRNPA1 in the starting material used for the immunoprecipitation.

Twenty micrograms of total protein from rat PC12 cells (lane 1) or PC12 cells expressing human FMRP (Tf hFMR1; lanes 2 and 3) that were cultured in the absence (lanes 1 and 2) or the presence of NGF for seven hours (lane 3). The western blot membrane was first reacted with anti-mRG (1:1500), the results of which are illustrated in the upper panel. The membrane was stripped and treated with anti-FMRP monoclonal antibody (1:5000) and is shown in the middle panel. The asterisk (*) indicates the migration site of human FMRP, which exhibits increased anti-mRG staining following NGF treatment. The membrane was stripped again and re-probed with anti-HSP70c, the constitutively expressed form of heat shock protein 70 as the protein loading control illustrated in the lower panel.