gst resin  (Elabscience Biotechnology)

Journal: Theranostics

Article Title: Discovery of a novel Nrf2 activator that modulates mitochondrial function in neurons by regulating DHRS3-Nrf2 interaction after ischemic stroke

doi: 10.7150/thno.128602

Figure Lengend Snippet: DHRS3 bound to Nrf2 and inhibited its binding to the ARE element, which was inhibited by Cpd.51. (A) Co-staining with DHRS3 and DAPI in SH-SY5Y cells demonstrated that OGD/R increased nuclear localization of DHRS3, which Cpd.51 effectively inhibited. (B) Three-dimensional structural models of DHRS3 and Nrf2 proteins, as well as the specific binding interfaces mediating their direct interaction, were generated using molecular docking approaches. (C, D) Immunoprecipitation assay of the interaction in whole cell lysate between Nrf2 and DHRS3 in physiological or OGD/R conditions, and SH-SY5Y cells treated with Cpd.51 (100 nM) or Vehicle (DMSO). n = 3. (E) Cpd.51 inhibited the interaction between Nrf2 and DHRS3 detected by GST pulldown. (F) DHRS3-overexpression inhibited the activation effect of Cpd.51 on the ARE luciferase reporter gene. n = 6. (G, H) DHRS3-overexpression suppressed the expression of downstream target gene and protein of Nrf2. n = 3 or 4. (I) The GST pull-down assay demonstrated that Nrf2 specifically bound to the region of DHRS3 encompassing amino acids 100 to 190. (J, K) Overexpression of DHRS3 inhibited the expression of downstream target genes and proteins of Nrf2, except for DHRS3 (1-100 aa). n = 3. (L) Three-dimensional and two-dimensional structural models of Cpd.51 and DHRS3 proteins, as well as the specific binding interfaces mediating their direct interaction, were generated using molecular docking approaches. (M) RMSD of complexes, proteins and small molecule ligands. (N) The distance between the binding sites of proteins and small molecules. (O) Free Energy Landscape. (P) SPR was employed to determinate of the binding affinity between Cpd.51 and DHRS3. (Q) CETSA analysis of intracellular binding between Cpd.51 (100 nM) and DHRS3 at different temperature. n = 3. Results are expressed as mean ± SD. J, ### P < 0.001 vs. NC-OE group. * P < 0.05, *** P < 0.001 vs. DHRS3-OE group. Statistical differences among groups were analyzed by using one-way ANOVA followed by Tukey's post-hoc test. G, ## P < 0.01, ### P < 0.001. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical differences among groups were analyzed by using two-way ANOVA followed by Tukey's post-hoc test.

Article Snippet: GST-Nrf2 (YB710012, Ybio, China) or GST (Ag0040, Proteintech, China, as control) proteins were mixed with GST Resin (EA-IP-K008, Elabscience Biotechnology, China) for 2 h incubation on the shaker at 4 °C.

Techniques: Binding Assay, Staining, Generated, Immunoprecipitation, Over Expression, Activation Assay, Luciferase, Expressing, Pull Down Assay

Journal: Molecular Plant Pathology

Article Title: The S s S mk1‐ S s S om1‐ S s M sb2 Pathway Regulates Infection Cushion Formation and Pathogenicity in Sclerotinia sclerotiorum

doi: 10.1111/mpp.70127

Figure Lengend Snippet: SsSmk1 is essential for infection cushion formation and interacts with SsSom1 in Sclerotinia sclerotiorum . (a, b) The Δ Sssmk1 mutant failed to develop infection cushions on (a) glass slides and (b) onion epidermis. Potato dextrose agar (PDA) plugs colonised with the wild type (WT) and Δ Sssmk1 were transferred to glass slides or onion epidermis and imaged at 24 h post‐inoculation (hpi). Scale bars = 200 μm. (c) Phylogenetic analysis of SsSom1 transcription factors. The dendrogram was constructed using MEGA 10 software. (d) Yeast two‐hybrid (Y2H) assay confirmed the interaction between SsSom1 and SsSmk1. (e) Subcellular co‐localisation of GFP‐SsSmk1 and RFP‐SsSom1 in the nucleus. Fluorescence signals were captured using Leica microsystems CMS Gmbh (Stellaris 5). Nuclei were stained with DAPI (1 μg/mL; C0065, Solarbio). (f) Pull‐down assays demonstrating the interaction between SsSmk1 and SsSom1. Purified GUS‐GST (negative control) or SsSom1‐GST proteins were incubated with GST beads (50 μL) for 1 h at 4°C, followed by co‐incubation with SsSmk1‐His fusion protein for 3 h. Eluates were resolved by SDS‐PAGE to detect interactions. (g) Expression level of SsSOM1 in hyphae (S0: mycelial growth of WT on PDA for 2 dpi, PDA covered with cellophane), sclerotia (S1: 4 days post‐inoculation [dpi]; S2: 6 dpi; S3: 12 dpi), infection cushion (IC) and infection stage (6 and 12 hpi). Different letters indicate statistically significant differences ( p < 0.01).

Article Snippet: SsSom1‐GST was incubated with GST (50 μL) resin (Sangon Biotech) for 1 h at 4°C and co‐incubated with SsSmk1‐His fusion protein for 3 h. GST resin was collected and washed with phosphate buffer more than five times; then, 2 × SDS loading buffer was added to the GST beads and boiled for 10 min, then SDS‐PAGE was used to detect the interaction.

Techniques: Infection, Mutagenesis, Construct, Software, Y2H Assay, Fluorescence, Staining, Purification, Negative Control, Incubation, SDS Page, Expressing