sentinel mutation kit  (New England Biolabs)

Journal: The Journal of Biological Chemistry

Article Title: The RING-finger domain of Arabidopsis RMR functions as an E3 ligase essential for post-Golgi trafficking

doi: 10.1016/j.jbc.2025.110721

Figure Lengend Snippet: I234Y substitution disturbed the E3 ligase activity of AtRMR1-RING. A , in vitro ubiquitination assay showing AtRMR1-RING is an E3 ligase. One microgram of GST-tagged AtRMR1-RING or GST were mixed with 0.1 μg Arabidopsis E1 ubiquitin–activating enzyme (AtUba1), 0.3 μg E2 ubiquitin–conjugating enzyme (AtUbc30), 2.5 μg ubiquitin (AtUb) in the presence of Mg 2+ and ATP at 30 °C, 30 min, and analyzed by Western blotting using ubiquitin (FK2; Enzo Life Sciences) and GST (Abcam) antibodies. B , how AtRMR1-RING ( cyan ) interacts with AtUbc30 ( gray ) was predicted by homology modeling based on the crystal structures of the human RNF38–UbcH5B–ubiquitin complex (Protein Data Bank [PDB] code: 4V3K ). The conserved Ile234 of AtRMR1-RING is located at the interface between AtRMR1-RING and AtUbc30. C , I234Y substitution disturbed the interaction between AtRMR1-RING and AtUbc30. The purified AtUbc30 (50 μM; input) was mixed with 50 μM wildtype, I234Y variant of GST-AtRMR1-RING, or GST in the presence of GSTrap Sepharose (GE Healthcare). After extensive washing, bound proteins were eluted with 20 mM reduced glutathione and analyzed by 15% SDS-PAGE with Coomassie blue staining. AtUbc30 was coeluted with wildtype GST-AtRMR1-RING but not with the I234Y variant nor the GST control. D , I234Y substitution greatly reduced the E3 ligase activity of AtRMR1-RING. Wildtype or I234Y variant of GST-AtRMR1-RING (1 μg) was incubated with AtUba1 (0.1 μg), AtUbc30 (0 μg, 0.3 μg, or 1.5 μg), and AtUb (2.5 μg) in the presence of Mg 2+ and ATP at 30 °C, 30 min. Polyubiquitination was detected by Western blotting using ubiquitin and GST antibodies. Polyubiquitination was evident in wildtype GST-AtRMR1-RING, but not in the I234Y variant, suggesting that I234Y substitution greatly reduced the E3 ligase activity. GST, glutathione- S -transferase.

Article Snippet: The I234Y mutation was introduced using Q5 site–directed mutagenesis kit (New England BioLabs) with primers listed in .

Techniques: Activity Assay, In Vitro, Ubiquitin Proteomics, Western Blot, Purification, Variant Assay, SDS Page, Staining, Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The RING-finger domain of Arabidopsis RMR functions as an E3 ligase essential for post-Golgi trafficking

doi: 10.1016/j.jbc.2025.110721

Figure Lengend Snippet: I234Y substitution resulted in Golgi retention of GFP-TMD-AtRMR1-CT in Arabidopsis protoplasts. Arabidopsis protoplasts were cotransfected with wildtype or I234Y mutant of GFP-TMD-AtRMR1-CT and the ( A ) Golgi (ManI-mRFP) or ( B ) TGN (mRFP-SYP61) markers before confocal imaging of transfected cells. The scale bar represents 10 μm. Colocalization of GFP-TMD-AtRMR1-CT and the ( C ) Golgi or ( D ) TGN markers in the enlarged images were quantified by Pearson's colocalization coefficient ( r P ) using the Costes’ automatic threshold. Data represent mean ± SEM (n = 3). The r P values for the I234Y mutant and ManI-mRFP was significantly higher than that of the wildtype. ( p < 0.001, two-tailed t test). CT, C-terminal; TGN, trans -Golgi network; TMD, transmembrane domain.

Article Snippet: The I234Y mutation was introduced using Q5 site–directed mutagenesis kit (New England BioLabs) with primers listed in .

Techniques: Mutagenesis, Imaging, Transfection, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The RING-finger domain of Arabidopsis RMR functions as an E3 ligase essential for post-Golgi trafficking

doi: 10.1016/j.jbc.2025.110721

Figure Lengend Snippet: The RING domains of AtRMR2–4 are E3 ligases. A , sequence alignment of the RING domains of AtRMR1–6, showing conservation of the C3H2C3 motif (indicated by triangles ) and Ile234 in AtRMR. B , I234Y substitution disrupted E3 ligase activity of AtRMR2-RING. In the E3 ligase activity assay, GST, GST-tagged AtRMR2-RING, or its I234Y variant were mixed with Arabidopsis E1 ubiquitin–activating enzyme (AtUba1), E2 ubiquitin–conjugating enzyme (AtUbc30), and ubiquitin in the presence of Mg 2+ and ATP at 30 °C, 30 min, and analyzed by Western blotting using ubiquitin (FK2; Enzo Life Sciences) and GST (Abcam) antibodies. In the presence of both E1 and E2, polyubiquitination was evident in wildtype GST-AtRMR2-RING but not in the I234Y variant. C and D , the E3 ligase assay was performed for GST-tagged AtRMR3-RING or AtRMR4-RING. In the presence of both E1 and E2, polyubiquitination was observed for GST-AtRMR3-RING and GST-AtRMR4-RING. GST, glutathione- S -transferase.

Article Snippet: The I234Y mutation was introduced using Q5 site–directed mutagenesis kit (New England BioLabs) with primers listed in .

Techniques: Sequencing, Activity Assay, Variant Assay, Ubiquitin Proteomics, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The RING-finger domain of Arabidopsis RMR functions as an E3 ligase essential for post-Golgi trafficking

doi: 10.1016/j.jbc.2025.110721

Figure Lengend Snippet: I234Y substitution resulted in Golgi retention of GFP-AtRMR2 in Arabidopsis protoplasts. Arabidopsis protoplasts were cotransfected with wildtype or I234Y mutant of GFP-AtRMR2 and the ( A ) Golgi (ManI-mRFP) or ( B ) TGN (mRFP-SYP61) markers before confocal imaging of transfected cells. The scale bar represents 10 μm. Colocalization of GFP-AtRMR2 and the ( C ) Golgi or ( D ) TGN markers in the enlarged images was quantified by Pearson's colocalization coefficient ( r P ) using the Costes’ automatic threshold. Data represent mean ± SEM (n = 3). The r P values for the I234Y mutant and ManI-mRFP were significantly higher than that of the wildtype ( p < 0.01, two-tailed t test). TGN, trans -Golgi network.

Article Snippet: The I234Y mutation was introduced using Q5 site–directed mutagenesis kit (New England BioLabs) with primers listed in .

Techniques: Mutagenesis, Imaging, Transfection, Two Tailed Test

Journal: Molecular Metabolism

Article Title: Inhibition of Unc119b improves insulin sensitivity through potentiation of Rac1 activation in skeletal muscle and brown adipose tissue

doi: 10.1016/j.molmet.2025.102230

Figure Lengend Snippet: Inhibition of Unc119b increases insulin-stimulated activation of Rac1. A. Western blot measurement of AKT and AS160 phosphorylation in C2C12 myoblasts transfected with scrambled or Unc119b siRNA and treated with vehicle, 100 nM insulin, 10 μM C59 or both for 10 min. B–C. Active Rac1 Western blot following pull down with PAK1-PDB coated beads in C2C12 myoblasts treated with vehicle, 100 nM insulin, 10 μM C59 or both for 5 min. Densitometry analysis shown in C. n = 6. D. G-LISA for Rac1 activity in C2C12 myoblasts treated with vehicle, 100 nM insulin, 10 μM C59 or both for 5 min, n = 8. F-G. Phospho-PAK1 Western blot from C2C12 myoblasts treated with vehicle, 100 nM insulin, 10 μM C59 or both for 5 min, n = 6. One-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Rac1 mutations were generated using Q5 site-directed mutagenesis kit (NEB) and verified by sequencing.

Techniques: Inhibition, Activation Assay, Western Blot, Phospho-proteomics, Transfection, Activity Assay

Journal: Molecular Metabolism

Article Title: Inhibition of Unc119b improves insulin sensitivity through potentiation of Rac1 activation in skeletal muscle and brown adipose tissue

doi: 10.1016/j.molmet.2025.102230

Figure Lengend Snippet: Interaction of Rac1 with Unc119b through the lipid moiety. A. Immunoprecipitation of Unc119b followed by Western blot detection of Rac1, Rac1-C178S, Rac1-C189S or Rac1-C178S/C189S. EV = Empty Vector. B. Structure of Unc119b in complex with geranylgeranylated cysteine (GG-Cys). The residues within 5 Å of the geranylgeranyl group are shown as sticks. One side of the binding pocket is lined by hydrophobic residues whereas the other side is lined with polar residues. Polder map around the geranylgeranyl group is shown at 2.5 sigma. C . Structure of Unc119b and GG-Cys showing hydrogen bonding interaction between the carboxy group of GG-Cys and Unc119b side chains. 2Fo-Fc map at 1 sigma is shown around the water mediating hydrogen bonding interaction between GG-Cys and Unc119b. D. Structural alignment between Unc119b-GG-Cys (green, GG-Cys orange sticks) and Unc119a-C59 (light grey, C59 – wheat sticks; PDB id. 7UMO) shows an overlapping binding site for the ligands. E. Schematic illustration of the BRET assay. F. BRET ratio measured in HEK293 cells transfected with EGFP-Rac1 and RLuc8-Unc119b after subtraction of background signal. t-test ∗∗∗p < 0.001.

Article Snippet: Rac1 mutations were generated using Q5 site-directed mutagenesis kit (NEB) and verified by sequencing.

Techniques: Immunoprecipitation, Western Blot, Plasmid Preparation, Binding Assay, Bioluminescence Resonance Energy Transfer, Transfection