Journal: Journal of Biological Chemistry

Article Title: Ran-binding Protein 3 Links Crm1 to the Ran Guanine Nucleotide Exchange Factor

doi: 10.1074/jbc.c100620200

Figure Lengend Snippet: FIG. 1. Ran stimulates binding of RCC1 to RanBP3. A, S. cerevi- siae W303 (MAT) expressing the VP16 transactivation domain alone or fused to Ran were mated with HF7c (MATa) transformed with the Gal4p DBD alone or fused to RCC1 or RCC1(D182A). Diploid yeast were replica plated onto LeuTrpHis. B, wild-type RanGDP or Ran(Q69L), a mutant Ran locked in the GTP-bound state (all 10 M), was incubated with GST or GST-RanBP3 bound to GSH beads. Bound and unbound proteins were subjected to SDS-PAGE and immunoblot- ting. C, RCC1 was incubated Ran(Q69L) or wild-type RanGDP with GST or GST-BP3 bound to GSH beads. Samples were analyzed as above. D, RCC1 Ran(Q69L) was incubated with GST, GST-BP3, GST-R (RanBP3 RBD), or GST-NF (RanBP3 minus the RBD) bound to beads. Samples were analyzed as above.

Article Snippet: Immunoblotting—RCC1 was visualized using an anti-RCC1 antibody (1:200; N-19, Santa Cruz Biotechnology) and a horseradish peroxidase-coupled rabbit anti-goat secondary antibody (1:20,000; The Jackson Laboratories).

Techniques: Binding Assay, Expressing, Transformation Assay, Mutagenesis, Incubation, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Ran-binding Protein 3 Links Crm1 to the Ran Guanine Nucleotide Exchange Factor

doi: 10.1074/jbc.c100620200

Figure Lengend Snippet: FIG. 2. RanBP3 enhances the catalytic activity of RCC1. A and B, Ran, loaded with [-32P]GTP or [-32P]GDP, was incubated with RCC1 (0.6 nM), RanBP3, and excess cold GDP or GTP for 3 min at 30 °C and then analyzed by binding to nitrocellulose and scintillation counting. Dissociation rate constants were calculated assuming a single exponential decay. Error bars represent the range from the mean of two experiments. C, as above except the exchange reaction was performed with RanBP3 or RanBP1 (5 M). The * indicates that RanBP1-treated Ran had a slower dissociation rate constant than Ran treated with buffer alone. Error bars represent 1 S.D. from the mean (n 3). D, exchange reactions were performed histones H2A/H2B (500 nM). Error bars represent the range from the mean of two experiments.

Article Snippet: Immunoblotting—RCC1 was visualized using an anti-RCC1 antibody (1:200; N-19, Santa Cruz Biotechnology) and a horseradish peroxidase-coupled rabbit anti-goat secondary antibody (1:20,000; The Jackson Laboratories).

Techniques: Activity Assay, Incubation, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Ran-binding Protein 3 Links Crm1 to the Ran Guanine Nucleotide Exchange Factor

doi: 10.1074/jbc.c100620200

Figure Lengend Snippet: FIG. 3. RanBP3 and Ran recruit Crm1 to RCC1. A and B, Crm1, RanBP3, and Ran were incubated with GST or GST-RCC1 bound to GSH beads. Proteins were analyzed as in Fig. 1. C, RCC1-catalyzed exchange reactions were performed RanBP3 (5 M) and Crm1 (5 M) as in Fig. 2. Error bars represent the range from the mean of two experiments.

Article Snippet: Immunoblotting—RCC1 was visualized using an anti-RCC1 antibody (1:200; N-19, Santa Cruz Biotechnology) and a horseradish peroxidase-coupled rabbit anti-goat secondary antibody (1:20,000; The Jackson Laboratories).

Techniques: Incubation

Journal: Journal of medicinal chemistry

Article Title: Selective Peptidomimetic Inhibitors of NTMT1/2: Rational design, synthesis, characterization, and crystallographic studies

doi: 10.1021/acs.jmedchem.0c00689

Figure Lengend Snippet: Inhibition mechanism of BM30. All experiments were run in triplicate (n=3). (A) IC50 curves of BM30 at varying concentrations of NTMT1 peptide substrate, RCC1–6 (SPKRIA), with fixed concentration of SAM; (B) linear regression plot of IC50 values with corresponding concentrations of RCC1–6; (C) IC50 curves of BM30 at varying concentrations of SAM with fixed concentration of RCC1–6; (D) Plot of IC50 values with corresponding concentrations of SAM.

Article Snippet: Antibodies against following proteins were used: Me3-SPK (Schaner Tooley lab 6 ), RCC1 (Proteintech, #22142–1-AP), SET (Proteintech, #55201–1-AP), Lamin B1 (Proteintech, # 12987–1-AP), Anti-rabbit IgG, HRP-linked (Cell Signaling, #7074S).

Techniques: Inhibition, Concentration Assay

Journal: Journal of medicinal chemistry

Article Title: Selective Peptidomimetic Inhibitors of NTMT1/2: Rational design, synthesis, characterization, and crystallographic studies

doi: 10.1021/acs.jmedchem.0c00689

Figure Lengend Snippet: Biochemical characterization of DC431 and DC432. (A,B) IC50 curve of DC431 and DC432. IC50 values were determined in triplicates (n = 3) and presented as mean ± SD. (C) Representative western blots showing N-terminal dimethylation (Me2RCC1) and trimethylation (Me3RCC1) of full-length recombinant human RCC1 by full-length recombinant human NTMT1 in the presence of increasing concentrations of BM30, DC431 and DC432 (n=3). NTMT1 and RCC1 blots are shown as loading controls.

Article Snippet: Antibodies against following proteins were used: Me3-SPK (Schaner Tooley lab 6 ), RCC1 (Proteintech, #22142–1-AP), SET (Proteintech, #55201–1-AP), Lamin B1 (Proteintech, # 12987–1-AP), Anti-rabbit IgG, HRP-linked (Cell Signaling, #7074S).

Techniques: Western Blot, Recombinant

Journal: Journal of medicinal chemistry

Article Title: Selective Peptidomimetic Inhibitors of NTMT1/2: Rational design, synthesis, characterization, and crystallographic studies

doi: 10.1021/acs.jmedchem.0c00689

Figure Lengend Snippet: Cellular N-terminal methylation assay of compound DC432 in HCT116 cells. (A) Structure of DC432. (B) Representative western blot results of effects of DC432 (0–1 mM) on cellular methylation level (n=2). RCC1, SET, and Lamin B1 blots are shown as loading controls.

Article Snippet: Antibodies against following proteins were used: Me3-SPK (Schaner Tooley lab 6 ), RCC1 (Proteintech, #22142–1-AP), SET (Proteintech, #55201–1-AP), Lamin B1 (Proteintech, # 12987–1-AP), Anti-rabbit IgG, HRP-linked (Cell Signaling, #7074S).

Techniques: Methylation, Western Blot