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Azenta dna coding sequences
Dna Coding Sequences, supplied by Azenta, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/dna+coding+sequence/coding+hnrnpk+sequences/pm41389987-101-0-19
Average 86 stars, based on 1 article reviews

dna coding sequences - by Bioz Stars, 2026-10

86/100 stars

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Expressing:

Article Title: Augmented prediction of multi-species protein-RNA interactions using evolutionary conservation of RNA-binding proteins.
Article Snippet: .. The myc-tagged zebrafish Tra2a and Hnrnpk coding sequences were codon-optimized for expression in HEK293T cells, synthesized by Azenta (Beijing, China), and PCRamplified using 2× Phanta Max Master Mix (Vazyme). ..

Article Title: Generation and NGS profiling of naïve rabbit antibody libraries for efficient monoclonal antibody selection.
Article Snippet: DNA coding sequences of the variable regions of the heavy and light chains from the selected scFvs were synthesized (GeneWiz, Leipzig, Germany) and cloned into pFUSE expression vectors (InvivoGen) containing the constant regions of rabbit IgG heavy chains (pFUSEss-CHIg-rG) and κ light chains (pFUSE2ss-CLIg-rk1 or pFUSE2ss-CLIg-rk2). .. DNA coding sequences of the variable regions of the heavy and light chains from the selected scFvs were synthesized (GeneWiz, Leipzig, Germany) and cloned into pFUSE expression vectors (InvivoGen) containing the constant regions of rabbit IgG heavy chains (pFUSEss-CHIg-rG) and κ light chains (pFUSE2ss-CLIg-rk1 or pFUSE2ss-CLIg-rk2). ..

Synthesized:

Article Title: Glycerol-3-phosphate activates ChREBP, FGF21 transcription and lipogenesis in citrin deficiency
Article Snippet: .. Human ChREBPα (accession number NM_032951.3 ), human ChREBPβ (accession number XM_047420437.1 ), human MLX (accession number NM_170607.3 ) and eGFP coding sequences were synthesized by Genewiz and cloned into pcDNA3.1 vectors (Invitrogen). .. GK was subcloned into pcDNA3.1 (Invitrogen) from pWZL-Neo-Myr-Flag-GK (Addgene plasmid 20493). pcDNA3.1-GPD1 and pcDNA3.1-GAPDH were purchased from GenScript (clone ID: OHu20325 and OHu20566).

Clone Assay:

Transfection:

Article Title: Harnessing design of experiments and automation significantly improves transient protein production in Chinese hamster ovary cells and accelerates drug discovery.
Article Snippet: .. For transfection, a Tecan Freedom EVO150 liquid handling workstation (Tecan Group Ltd., Switzerland) was utilized to dispense the coding DNA plasmids (Genewiz), PEIMax transfection reagent (Polysciences Inc., Cat. 24765–1, pre-diluted to 1 mg/mL in deionized water), and, if applicable, filler DNA at 10 mg/mL and ranged in size from 200 to 500 bp (salmon sperm DNA, Invitrogen, Cat. AM9680). .. This Tecan workstation is configured with an eight-channel liquid handling (LiHa) system with disposable tips (DiTi), a customized pick and place (PnP) arm for the de-/capping of the 50-mL tube reactors, a high-efficiency particulate air (HEPA) filter hood, and an integrated Shaker (Ika, KS260, 10 mm orbit, cat. 0002980301) for the 48-positioned tubespin rack.

Image Search Results

Donor mito-mTagBFP2 can be free from endocytic vesicles. HUVECs expressing cell surface GFP and endosome-targeted Rab5a-TagRFP (magenta) transplanted with mito-mTagBFP2 displaying anti-GFP nanobody (cyan). The videos were recorded 6 h and 1 day after mitochondrial transplantation, respectively. Two different cells are shown. Scale bar, 5 mm.

Journal: Nature

Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration

doi: 10.1038/s41586-026-10391-0

Figure Lengend Snippet: Donor mito-mTagBFP2 can be free from endocytic vesicles. HUVECs expressing cell surface GFP and endosome-targeted Rab5a-TagRFP (magenta) transplanted with mito-mTagBFP2 displaying anti-GFP nanobody (cyan). The videos were recorded 6 h and 1 day after mitochondrial transplantation, respectively. Two different cells are shown. Scale bar, 5 mm.

Article Snippet: For TagBFP2 targeting into the matrix, the mTagBFP2 coding DNA sequence was fused to COX8 matrix-targeting signal peptide, synthesized by Twist Biosciences, and inserted into a pCMV backbone.

Techniques:

a. Top, schematic diagram of the construct used for directing a nanobody to the outer membrane of mitochondria. Bottom, a super-resolution image of an HEK293T cell with mitochondria-targeted nanobody detected by anti-alpacaV H H antibodies (magenta). Cell nuclei are labelled with Hoechst (blue). Mitochondria matrix is labelled with dsRed2 (cyan). 3D SIM, three-dimensional structural illumination microscopy. The construct was validated in at least three independent experiments. b. Top, schematic diagram of the construct used for directing GFP to the cell surface. Bottom, a super-resolution image of an HEK293T cell with cell surface-targeted GFP detected by anti-GFP antibodies (green). Cell nuclei are labelled with Hoechst (blue). The construct was validated in at least three independent experiments. c. HEK239T cells transplanted with donor mitochondria displaying the anti-GFP nanobody, two hours after transplantation. HEK293T cells are transfected with cell-surface mCherry (cyan) or GFP (green). Nanobodies are detected by anti-alpacaV H H antibodies (magenta). d. Quantification of the efficacy of the delivery of nanobody-displaying mitochondria two hours after transplantation. n = 6, P < 0.0001 (top) and P = 0.0012 (bottom), two-sided Welch’s t test. e. HEK239T cells transplanted with donor mitochondria displaying the anti-mCherry nanobody, two hours after transplantation. HEK293T cells were transfected with cell-surface GFP (green) or mCherry (cyan). Nanobodies detected by anti-alpacaV H H antibodies (magenta). f. Quantification of the efficacy of the delivery of nanobody-displaying mitochondria, two hours after transplantation. n = 8, P = 0.0003 (top) and P = 0.0012 (bottom), two-sided Welch’s test. g. Live-imaged endothelial cells expressing cell surface GFP and endosome-targeted RAB5A-TagRFP with (top) and without (bottom) transplanted with mito-mTagBFP2 (cyan) displaying anti-GFP nanobody, six hours after mitochondrial transplantation. White arrows, endosome-free donor mitochondria (confirmed in at least three independent experiments). For an example of mito-mTagBP2 in endothelial cells transplanted without the anti-GFP nanobody, see Supplementary Fig. . h. Donor mito-mTagBFP2 (cyan) inside endocytic vesicles (red arrows) labelled with RAB5A-TagRFP (magenta) (from g), six hours after mitochondrial transplantation. i. Donor mito-mTagBFP2 (cyan) free from endocytic vesicles (white arrows) labelled with RAB5A-TagRFP (magenta) (from g), six hours after mitochondrial transplantation. j. Donor mito-mTagBFP2 (cyan) free from endocytic vesicles (white arrows) labelled with RAB5A-TagRFP (magenta), 24 h after mitochondrial transplantation. Outlined region, mito-mTagBFP2 free from endocytic vesicles and lysosomes (yellow). Lysosomes are stained with LysoTracker Deep Red dye. The experiment was repeated at least three times with similar results. k. Quantification of proportion of endosome-free mitochondria by pixel-based co-localization analysis. Mito-mTagBFP2: n = 30; mito-mTagBFP2 + anti-GFP nanobody: n = 52, P = 0.0756, two-sided Mann-Whitney U test. l. Quantification of abundance of endosome-free mitochondria by pixel-based co-localization analysis. The values were normalized to cell size (µm 2 of donor mitochondria area per 1 µm 2 cell area). Mito-mTagBFP2: n = 30; mito-mTagBFP2 + anti-GFP nanobody: n = 52, P < 0.0001, two-sided Mann-Whitney U test. m. Endothelial cells stained with pH-dependent lysosome staining dye pHLys Red (yellow). Cells expressed cell surface GFP and were transplanted with mito-mTagBFP2 displaying anti-GFP nanobody or no binder. In addition, Bafilomycin A1 was used as a positive control for pH acidification change in lysosomes. For mitochondria transplanted conditions, images of cells positive for mito-mTagBFP2 are shown (Supplementary Fig. ). n. Quantification of pH changes in lysosomes relative to untreated condition. Untreated: n = 6; Bafilomycin A1: n = 4; mito-mTagBFP2: n = 4; mito-mTagBFP2 + anti-GFP nanobody: n = 6, Untreated vs. Bafilomycin A1: P = 0.0036, Untreated vs. mito-mTagBFP2: P = 0.8235, Untreated vs. mito-mTagBFP2 + anti-GFP nanobody: P = 0.9648, Welch’s ANOVA test corrected with two-sided Dunnett’s test for multiple comparisons. o. Live-imaged endothelial cell expressing cell surface GFP (green), and transplanted with mito-dsRed2 (cyan) displaying anti-GFP nanobody, four days after mitochondrial transplantation. The cell is outlined with a grey dashed line. The zoomed-in region is outlined with a white dashed square. Two timeframes are shown on the right. The tracked mitochondrion is indicated with a red arrow. The experiment was repeated at least three times with similar results. p. Live-imaged endothelial cell expressing cell surface GFP and transplanted with mito-dsRed2 (cyan) displaying outer membrane anti-GFP nanobody, four days after mitochondrial transplantation. Mitochondria are labelled with 50 nM MitoTracker Deep Red (magenta). The zoomed-in region is outlined with a white dashed square and the tracked mitochondrion is indicated with a white arrow. The experiment was repeated at least three times with similar results. q. Labelling of donor and native mitochondria with MitoTracker Deep Red dye in live-recorded endothelial cells. At the used concentration, the dye stained both native (black) and donor mitochondria (cyan) with stronger enrichment in the native mitochondria. Donor mitochondria positive for matrix-labelled dsRed2 and MitoTracker Deep Red are indicated with red arrows. NS not significant, ** P < 0.01, *** P < 0.001. Data, mean ± s.e.m and median for k, l. Scale bars, 2.5 µm (a, b), 25 µm (c, e), 5 µm (g, h, i, o, p, q), 10 µm (j), 20 µm (m).

Journal: Nature

Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration

doi: 10.1038/s41586-026-10391-0

Figure Lengend Snippet: a. Top, schematic diagram of the construct used for directing a nanobody to the outer membrane of mitochondria. Bottom, a super-resolution image of an HEK293T cell with mitochondria-targeted nanobody detected by anti-alpacaV H H antibodies (magenta). Cell nuclei are labelled with Hoechst (blue). Mitochondria matrix is labelled with dsRed2 (cyan). 3D SIM, three-dimensional structural illumination microscopy. The construct was validated in at least three independent experiments. b. Top, schematic diagram of the construct used for directing GFP to the cell surface. Bottom, a super-resolution image of an HEK293T cell with cell surface-targeted GFP detected by anti-GFP antibodies (green). Cell nuclei are labelled with Hoechst (blue). The construct was validated in at least three independent experiments. c. HEK239T cells transplanted with donor mitochondria displaying the anti-GFP nanobody, two hours after transplantation. HEK293T cells are transfected with cell-surface mCherry (cyan) or GFP (green). Nanobodies are detected by anti-alpacaV H H antibodies (magenta). d. Quantification of the efficacy of the delivery of nanobody-displaying mitochondria two hours after transplantation. n = 6, P < 0.0001 (top) and P = 0.0012 (bottom), two-sided Welch’s t test. e. HEK239T cells transplanted with donor mitochondria displaying the anti-mCherry nanobody, two hours after transplantation. HEK293T cells were transfected with cell-surface GFP (green) or mCherry (cyan). Nanobodies detected by anti-alpacaV H H antibodies (magenta). f. Quantification of the efficacy of the delivery of nanobody-displaying mitochondria, two hours after transplantation. n = 8, P = 0.0003 (top) and P = 0.0012 (bottom), two-sided Welch’s test. g. Live-imaged endothelial cells expressing cell surface GFP and endosome-targeted RAB5A-TagRFP with (top) and without (bottom) transplanted with mito-mTagBFP2 (cyan) displaying anti-GFP nanobody, six hours after mitochondrial transplantation. White arrows, endosome-free donor mitochondria (confirmed in at least three independent experiments). For an example of mito-mTagBP2 in endothelial cells transplanted without the anti-GFP nanobody, see Supplementary Fig. . h. Donor mito-mTagBFP2 (cyan) inside endocytic vesicles (red arrows) labelled with RAB5A-TagRFP (magenta) (from g), six hours after mitochondrial transplantation. i. Donor mito-mTagBFP2 (cyan) free from endocytic vesicles (white arrows) labelled with RAB5A-TagRFP (magenta) (from g), six hours after mitochondrial transplantation. j. Donor mito-mTagBFP2 (cyan) free from endocytic vesicles (white arrows) labelled with RAB5A-TagRFP (magenta), 24 h after mitochondrial transplantation. Outlined region, mito-mTagBFP2 free from endocytic vesicles and lysosomes (yellow). Lysosomes are stained with LysoTracker Deep Red dye. The experiment was repeated at least three times with similar results. k. Quantification of proportion of endosome-free mitochondria by pixel-based co-localization analysis. Mito-mTagBFP2: n = 30; mito-mTagBFP2 + anti-GFP nanobody: n = 52, P = 0.0756, two-sided Mann-Whitney U test. l. Quantification of abundance of endosome-free mitochondria by pixel-based co-localization analysis. The values were normalized to cell size (µm 2 of donor mitochondria area per 1 µm 2 cell area). Mito-mTagBFP2: n = 30; mito-mTagBFP2 + anti-GFP nanobody: n = 52, P < 0.0001, two-sided Mann-Whitney U test. m. Endothelial cells stained with pH-dependent lysosome staining dye pHLys Red (yellow). Cells expressed cell surface GFP and were transplanted with mito-mTagBFP2 displaying anti-GFP nanobody or no binder. In addition, Bafilomycin A1 was used as a positive control for pH acidification change in lysosomes. For mitochondria transplanted conditions, images of cells positive for mito-mTagBFP2 are shown (Supplementary Fig. ). n. Quantification of pH changes in lysosomes relative to untreated condition. Untreated: n = 6; Bafilomycin A1: n = 4; mito-mTagBFP2: n = 4; mito-mTagBFP2 + anti-GFP nanobody: n = 6, Untreated vs. Bafilomycin A1: P = 0.0036, Untreated vs. mito-mTagBFP2: P = 0.8235, Untreated vs. mito-mTagBFP2 + anti-GFP nanobody: P = 0.9648, Welch’s ANOVA test corrected with two-sided Dunnett’s test for multiple comparisons. o. Live-imaged endothelial cell expressing cell surface GFP (green), and transplanted with mito-dsRed2 (cyan) displaying anti-GFP nanobody, four days after mitochondrial transplantation. The cell is outlined with a grey dashed line. The zoomed-in region is outlined with a white dashed square. Two timeframes are shown on the right. The tracked mitochondrion is indicated with a red arrow. The experiment was repeated at least three times with similar results. p. Live-imaged endothelial cell expressing cell surface GFP and transplanted with mito-dsRed2 (cyan) displaying outer membrane anti-GFP nanobody, four days after mitochondrial transplantation. Mitochondria are labelled with 50 nM MitoTracker Deep Red (magenta). The zoomed-in region is outlined with a white dashed square and the tracked mitochondrion is indicated with a white arrow. The experiment was repeated at least three times with similar results. q. Labelling of donor and native mitochondria with MitoTracker Deep Red dye in live-recorded endothelial cells. At the used concentration, the dye stained both native (black) and donor mitochondria (cyan) with stronger enrichment in the native mitochondria. Donor mitochondria positive for matrix-labelled dsRed2 and MitoTracker Deep Red are indicated with red arrows. NS not significant, ** P < 0.01, *** P < 0.001. Data, mean ± s.e.m and median for k, l. Scale bars, 2.5 µm (a, b), 25 µm (c, e), 5 µm (g, h, i, o, p, q), 10 µm (j), 20 µm (m).

Techniques: Construct, Membrane, Microscopy, Transplantation Assay, Transfection, Expressing, Staining, MANN-WHITNEY, Positive Control, Concentration Assay

Silencing of GmMEKK2 by virus‐induced gene silencing (VIGS) increased soybean mosaic virus (SMV) susceptibility. (A) Efficiency of GmMEKK2 silencing in empty vector control (EV) and GmMEKK2 ‐silenced mekk2 i1 and mekk2 i2 plants at 0, 7, 14 and 21 days post‐inoculation (dpi). (B) Phenotypes of soybean after SMV infection: EV and GmMEKK2 ‐silenced lines generated using VIGS. Images were taken at 21 dpi. (C) Disease indices of plants at 21 dpi. Lowercase letters denote statistically significant differences among groups at the same time point ( p < 0.05, one‐way ANOVA with Duncan's test). (D) Relative SMV accumulation in top new leaves of EV and GmMEKK2 ‐silenced plants at 7, 14 and 21 dpi, quantified by reverse transcription‐quantitative PCR using SMV coat protein‐specific primers.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Silencing of GmMEKK2 by virus‐induced gene silencing (VIGS) increased soybean mosaic virus (SMV) susceptibility. (A) Efficiency of GmMEKK2 silencing in empty vector control (EV) and GmMEKK2 ‐silenced mekk2 i1 and mekk2 i2 plants at 0, 7, 14 and 21 days post‐inoculation (dpi). (B) Phenotypes of soybean after SMV infection: EV and GmMEKK2 ‐silenced lines generated using VIGS. Images were taken at 21 dpi. (C) Disease indices of plants at 21 dpi. Lowercase letters denote statistically significant differences among groups at the same time point ( p < 0.05, one‐way ANOVA with Duncan's test). (D) Relative SMV accumulation in top new leaves of EV and GmMEKK2 ‐silenced plants at 7, 14 and 21 dpi, quantified by reverse transcription‐quantitative PCR using SMV coat protein‐specific primers.

Article Snippet: The GmMEKK2 full‐length coding DNA sequence (CDS) was inserted into pDONOR221 (Invitrogen) and then transferred to a pB7FWG2 vector via an LR recombination reaction in the Gateway system.

Techniques: Virus, Plasmid Preparation, Control, Infection, Generated, Reverse Transcription, Real-time Polymerase Chain Reaction

Overexpression of GmMEKK2 improved soybean mosaic virus (SMV) resistance in soybean. (A) Infection symptoms on soybean leaves after SMV inoculation. NT, nontransgenic plants; ZMP1, 3, 6 and 7 indicate GmMEKK2 ‐overexpression lines 1, 3, 6 and 7, respectively. (B) Disease indices of NT and each GmMEKK2 ‐overexpression line. The disease index was investigated at 21 days post‐SMV‐inoculation. (C) Quantification of SMV content in soybean leaves. SMV‐susceptible line 1138‐2 was used as a positive control. (D) The GmMEKK2 expression pattern in NT plants after SMV inoculation. (E) Comparison of yield traits between NT and overexpression plants after SMV infection. Mock‐inoculated plants served as the control. Values labelled with different lowercase letters (a–e) are significantly different at p < 0.05 as determined by Duncan's test.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Overexpression of GmMEKK2 improved soybean mosaic virus (SMV) resistance in soybean. (A) Infection symptoms on soybean leaves after SMV inoculation. NT, nontransgenic plants; ZMP1, 3, 6 and 7 indicate GmMEKK2 ‐overexpression lines 1, 3, 6 and 7, respectively. (B) Disease indices of NT and each GmMEKK2 ‐overexpression line. The disease index was investigated at 21 days post‐SMV‐inoculation. (C) Quantification of SMV content in soybean leaves. SMV‐susceptible line 1138‐2 was used as a positive control. (D) The GmMEKK2 expression pattern in NT plants after SMV inoculation. (E) Comparison of yield traits between NT and overexpression plants after SMV infection. Mock‐inoculated plants served as the control. Values labelled with different lowercase letters (a–e) are significantly different at p < 0.05 as determined by Duncan's test.

Techniques: Over Expression, Virus, Infection, Positive Control, Expressing, Comparison, Control

Expression profiles of key differentially expressed genes (DEGs) between nontransgenic (NT) and GmMEKK2 ‐overexpression lines (ZMP) involved in the reactive oxygen species (ROS)‐ and salicylic acid (SA)‐related pathways. (A) KEGG enrichment analysis of DEGs between NT and ZMP plants. Left: NT_CK versus ZMP_CK (uninfected controls); Right: NT_7d versus ZMP_7d (7 days post‐SMV‐inoculation [dpi]). Points represent enriched pathways, with size indicating gene count and colour reflecting −log 10 (adjusted p ‐value). Red arrows highlight defence‐related pathways. (B) Expression dynamics of key components among MAPK, plant hormone signalling and plant–pathogen interaction pathways. Schematic depicts signal transduction from apoplast to cytoplasm, including Ca 2+ sensors (CNGCs and CDPKs), ROS producers (Rbohs) and SA‐induced defence protein (PR1). Heatmaps show expression levels across conditions (NT and ZMP at 0, 7 and 14 dpi), with gene IDs labelled.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Expression profiles of key differentially expressed genes (DEGs) between nontransgenic (NT) and GmMEKK2 ‐overexpression lines (ZMP) involved in the reactive oxygen species (ROS)‐ and salicylic acid (SA)‐related pathways. (A) KEGG enrichment analysis of DEGs between NT and ZMP plants. Left: NT_CK versus ZMP_CK (uninfected controls); Right: NT_7d versus ZMP_7d (7 days post‐SMV‐inoculation [dpi]). Points represent enriched pathways, with size indicating gene count and colour reflecting −log 10 (adjusted p ‐value). Red arrows highlight defence‐related pathways. (B) Expression dynamics of key components among MAPK, plant hormone signalling and plant–pathogen interaction pathways. Schematic depicts signal transduction from apoplast to cytoplasm, including Ca 2+ sensors (CNGCs and CDPKs), ROS producers (Rbohs) and SA‐induced defence protein (PR1). Heatmaps show expression levels across conditions (NT and ZMP at 0, 7 and 14 dpi), with gene IDs labelled.

Techniques: Expressing, Over Expression, Transduction

Kinase activity of GmMEKK2 is dispensable for its function in mediating defence signalling. (A–E) Relative expression levels of (A) GmMKK1 , (B) GmMPK4A , (C) GmMPK13‐like , (D) GmSUMM2 and (E) GmCRCK3 in nontransgenic control (NT), GmMEKK2 ‐overexpression lines (ZMP1, ZMP3 and ZMP7), empty vector control (EV) and GmMEKK2‐ silenced lines ( mekk2 i1 and mekk2 i2 ). Lowercase letters denote significant differences at p < 0.05 as determined by one‐way ANOVA with Duncan's test. (F) Domain architecture of GmMEKK2 highlighting the kinase domain (6–264 amino acids) and ATP‐binding site (K36). Autophosphorylation of GmMEKK2 was assessed by immunoblotting with α‐pSer/Thr antibody. Recombinant proteins GmMEKK1‐FLAG and GmMEKK1 K321M ‐FLAG were used as positive and negative controls, respectively. Coomassie brilliant blue staining validated the equal loading of recombinant proteins. (G) Yeast two‐hybrid analysis of GmMEKK2 interaction with GmMKK1, GmMPK4A and GmMPK13‐like. Transformants expressing pGADT7 and pGBKT7 constructs were grown on SD/−Leu/−Trp (control) and SD/−Leu/−Trp/−Ade/−His (selection) media. (H–J) Glutathione S‐transferase (GST) pull‐down assays with anti‐His and anti‐GST antibodies demonstrating direct binding between GST‐GmMEKK2 and (H) GmMKK1‐His, (I) GmMPK4A‐His and (J) GmMPK13‐like‐His.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Kinase activity of GmMEKK2 is dispensable for its function in mediating defence signalling. (A–E) Relative expression levels of (A) GmMKK1 , (B) GmMPK4A , (C) GmMPK13‐like , (D) GmSUMM2 and (E) GmCRCK3 in nontransgenic control (NT), GmMEKK2 ‐overexpression lines (ZMP1, ZMP3 and ZMP7), empty vector control (EV) and GmMEKK2‐ silenced lines ( mekk2 i1 and mekk2 i2 ). Lowercase letters denote significant differences at p < 0.05 as determined by one‐way ANOVA with Duncan's test. (F) Domain architecture of GmMEKK2 highlighting the kinase domain (6–264 amino acids) and ATP‐binding site (K36). Autophosphorylation of GmMEKK2 was assessed by immunoblotting with α‐pSer/Thr antibody. Recombinant proteins GmMEKK1‐FLAG and GmMEKK1 K321M ‐FLAG were used as positive and negative controls, respectively. Coomassie brilliant blue staining validated the equal loading of recombinant proteins. (G) Yeast two‐hybrid analysis of GmMEKK2 interaction with GmMKK1, GmMPK4A and GmMPK13‐like. Transformants expressing pGADT7 and pGBKT7 constructs were grown on SD/−Leu/−Trp (control) and SD/−Leu/−Trp/−Ade/−His (selection) media. (H–J) Glutathione S‐transferase (GST) pull‐down assays with anti‐His and anti‐GST antibodies demonstrating direct binding between GST‐GmMEKK2 and (H) GmMKK1‐His, (I) GmMPK4A‐His and (J) GmMPK13‐like‐His.

Techniques: Activity Assay, Expressing, Control, Over Expression, Plasmid Preparation, Binding Assay, Western Blot, Recombinant, Staining, Construct, Selection

GmMEKK2 promotes the immune response induced by salicylic acid (SA). (A) Contents of free (SA) and bound salicylic acid (SAG) in nontransgenic (NT) and GmMEKK2 ‐overexpression (ZMP) lines. (B) GmMEKK2 expression in NT plants after exogenous hormone treatments. ETH, ethylene; ABA, abscisic acid (C–H) Expression of pivotal genes in the SA signalling pathway in NT, GmMEKK2 ‐overexpression and GmMEKK2 ‐silenced ( mekk2 i1 and mekk2 i2 ) plants at 7 days post‐inoculation. EV, empty vector. Values labelled with different lowercase letters (a–c) are significantly different at p < 0.05 as determined by Duncan's test.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: GmMEKK2 promotes the immune response induced by salicylic acid (SA). (A) Contents of free (SA) and bound salicylic acid (SAG) in nontransgenic (NT) and GmMEKK2 ‐overexpression (ZMP) lines. (B) GmMEKK2 expression in NT plants after exogenous hormone treatments. ETH, ethylene; ABA, abscisic acid (C–H) Expression of pivotal genes in the SA signalling pathway in NT, GmMEKK2 ‐overexpression and GmMEKK2 ‐silenced ( mekk2 i1 and mekk2 i2 ) plants at 7 days post‐inoculation. EV, empty vector. Values labelled with different lowercase letters (a–c) are significantly different at p < 0.05 as determined by Duncan's test.

Techniques: Over Expression, Expressing, Plasmid Preparation

GmMEKK2 is involved in the regulation of reactive oxygen species homeostasis in soybean. (A, B) H 2 O 2 and O 2− levels in leaves were detected at 7 days post‐inoculation (dpi) using 3,3′‐diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, respectively. The mock‐inoculated leaves were sampled as controls. (C–G) Trends in the gene expression of antioxidases were measured after soybean mosaic virus (SMV) infection. CK, noninoculated control (H–J) Antioxidase activities were measured. POD, peroxidase; CAT, catalase; SOD, superoxide dismutase. The statistical analysis was independently performed for GmMEKK2 ‐overexpression lines ZMP1, ZMP3 and ZMP7, and gene‐silenced lines mekk2 i1 , mekk2 i2 and nontransgenic (NT) plants at each stage. Values labelled with different lowercase letters are significantly different at p < 0.05 as determined by Duncan's test.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: GmMEKK2 is involved in the regulation of reactive oxygen species homeostasis in soybean. (A, B) H 2 O 2 and O 2− levels in leaves were detected at 7 days post‐inoculation (dpi) using 3,3′‐diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, respectively. The mock‐inoculated leaves were sampled as controls. (C–G) Trends in the gene expression of antioxidases were measured after soybean mosaic virus (SMV) infection. CK, noninoculated control (H–J) Antioxidase activities were measured. POD, peroxidase; CAT, catalase; SOD, superoxide dismutase. The statistical analysis was independently performed for GmMEKK2 ‐overexpression lines ZMP1, ZMP3 and ZMP7, and gene‐silenced lines mekk2 i1 , mekk2 i2 and nontransgenic (NT) plants at each stage. Values labelled with different lowercase letters are significantly different at p < 0.05 as determined by Duncan's test.

Techniques: Staining, Gene Expression, Virus, Infection, Control, Over Expression

Molecular mechanisms underlying the GmMEKK2‐mediated regulation of soybean mosaic virus (SMV) resistance in soybean. (A) Phenotype and regulatory mechanism of GmMEKK2 ‐overexpression plants under SMV inoculation. Left: GmMEKK2 ‐overexpression plants (ZMP) show no visible SMV symptoms with autoimmunity phenotype such as leaf yellowing. Right: In ZMP plants, GmMEKK2 (orange ellipses) interacts with GmMKK1 and GmMPK4A, blocking the phosphorylation (letter P in a blue circle) of the GmMEKK1‐GmMKK1‐GmMPK4A cascade. This inhibition represses (cross in a red circle) WRKY transcription factors and leads to non‐phosphorylated CRCK3 releasing SUMM2. This then triggers defence responses such as salicylic acid (SA)‐induced gene expression and basal reactive oxygen species (ROS) accumulation. The elevated ROS constitutivly results in autoimmunity in ZMP plants. (B) Left: Nontransgenic (NT) plants exhibit severe SMV symptoms such as mosaic leaves and mottled pods. Right: In NT plants, GmMEKK2 expression is low, so the GmMEKK1‐GmMKK1‐GmMPK4A cascade remains active. GmMPK4A phosphorylates CRCK3, which binds with and represses SUMM2. This suppresses defence responses, and leads to a ROS burst.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Molecular mechanisms underlying the GmMEKK2‐mediated regulation of soybean mosaic virus (SMV) resistance in soybean. (A) Phenotype and regulatory mechanism of GmMEKK2 ‐overexpression plants under SMV inoculation. Left: GmMEKK2 ‐overexpression plants (ZMP) show no visible SMV symptoms with autoimmunity phenotype such as leaf yellowing. Right: In ZMP plants, GmMEKK2 (orange ellipses) interacts with GmMKK1 and GmMPK4A, blocking the phosphorylation (letter P in a blue circle) of the GmMEKK1‐GmMKK1‐GmMPK4A cascade. This inhibition represses (cross in a red circle) WRKY transcription factors and leads to non‐phosphorylated CRCK3 releasing SUMM2. This then triggers defence responses such as salicylic acid (SA)‐induced gene expression and basal reactive oxygen species (ROS) accumulation. The elevated ROS constitutivly results in autoimmunity in ZMP plants. (B) Left: Nontransgenic (NT) plants exhibit severe SMV symptoms such as mosaic leaves and mottled pods. Right: In NT plants, GmMEKK2 expression is low, so the GmMEKK1‐GmMKK1‐GmMPK4A cascade remains active. GmMPK4A phosphorylates CRCK3, which binds with and represses SUMM2. This suppresses defence responses, and leads to a ROS burst.

Techniques: Virus, Over Expression, Blocking Assay, Phospho-proteomics, Inhibition, Gene Expression, Expressing

a . Diagram of key molecules in the chemotaxis network. Extracellular ligands (black semicircles) bind to receptors and modulate the activity of CheA, the kinase that phosphorylates the diffusible messenger CheY. CheA activity adaptation is mediated by the methylase CheR and de-methylase CheB. CheY-P binds to the C-ring and triggers a conformational change that results in BFM changing direction (CCW → CW). The swimming bacterium switches from a smooth run to a tumble. b . AlphaFold structure predictions of chimeric molecules. Grey is E. coli CheY, blue is cpAsLOV2, black is a short linker, and orange is the FliM peptide. Cartoons depicting the “caged” dark-state of Opto-CheY and the constitutively active control are shown with the same color code. c . Blue-light absorption triggers unfolding of the J α helix, and CheY activation by removal of the FliM plug from the CheY active site. Active CheY binds to the C-ring and promotes the rotational switch of the BFM from CCW → CW. Light pulses (blue dots, ~100 µ J/cm 2 ) are applied every 6 seconds while monitoring BFM rotational state. Each response is a CW rotation (vertical black line) following a blue light pulse (blue dot) in an otherwise mostly CCW motor (bottom trace)

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: a . Diagram of key molecules in the chemotaxis network. Extracellular ligands (black semicircles) bind to receptors and modulate the activity of CheA, the kinase that phosphorylates the diffusible messenger CheY. CheA activity adaptation is mediated by the methylase CheR and de-methylase CheB. CheY-P binds to the C-ring and triggers a conformational change that results in BFM changing direction (CCW → CW). The swimming bacterium switches from a smooth run to a tumble. b . AlphaFold structure predictions of chimeric molecules. Grey is E. coli CheY, blue is cpAsLOV2, black is a short linker, and orange is the FliM peptide. Cartoons depicting the “caged” dark-state of Opto-CheY and the constitutively active control are shown with the same color code. c . Blue-light absorption triggers unfolding of the J α helix, and CheY activation by removal of the FliM plug from the CheY active site. Active CheY binds to the C-ring and promotes the rotational switch of the BFM from CCW → CW. Light pulses (blue dots, ~100 µ J/cm 2 ) are applied every 6 seconds while monitoring BFM rotational state. Each response is a CW rotation (vertical black line) following a blue light pulse (blue dot) in an otherwise mostly CCW motor (bottom trace)

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Chemotaxis Assay, Activity Assay, Control, Activation Assay

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet:

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Transformation Assay, Expressing, Variant Assay

Blue light pulses (2 msec, ~100 µ J/cm 2 ) are applied every 6 seconds. Results are shown from 3 different strains: 657 (no plasmid), 638 , and 658 (CheY-cpAsLOV2 N449S ). Binary traces of cell body rotation (CCW=0, CW=1) are shown as a running average of 100 frames (500 msec). Blue light pulses are shown as vertical blue lines.

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: Blue light pulses (2 msec, ~100 µ J/cm 2 ) are applied every 6 seconds. Results are shown from 3 different strains: 657 (no plasmid), 638 , and 658 (CheY-cpAsLOV2 N449S ). Binary traces of cell body rotation (CCW=0, CW=1) are shown as a running average of 100 frames (500 msec). Blue light pulses are shown as vertical blue lines.

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Plasmid Preparation

Averaged binary traces (100 frames running average, 500 msec) for strain 636 (Opto-CheY V416T ). Each plot is a single motor trace. Blue light pulses are shown as blue lines. 2 msec blue light pulses of a given blue light intensity ( I = I min · x, x = {1, 2, 4, 8, 16, 32}) are applied at constant frequency: every 4 sec for x = 1, every 6 sec for x = 2, every 8 sec for x = 4, every 10 sec for x = 8, every 12 sec for x = 16, and every 15 sec for x = 32.

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: Averaged binary traces (100 frames running average, 500 msec) for strain 636 (Opto-CheY V416T ). Each plot is a single motor trace. Blue light pulses are shown as blue lines. 2 msec blue light pulses of a given blue light intensity ( I = I min · x, x = {1, 2, 4, 8, 16, 32}) are applied at constant frequency: every 4 sec for x = 1, every 6 sec for x = 2, every 8 sec for x = 4, every 10 sec for x = 8, every 12 sec for x = 16, and every 15 sec for x = 32.

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques:

a . Opto-CheY V416T -driven motor responses. Each motor is flashed with a series of pulses of increasing relative intensity ( I = I min · x, x = {1, 2, 4, 8, 16, 32}, I min delivers ~ 7 · 10 12 blue photons/cm 2 ). Response probability for each BFM at a given intensity is calculated as the number of responses divided by the number of flashes. P see with θ = 2 is used to fit each dose response curve . Ten individual BFM response curves and fits are shown translated horizontally relative to half-maximum response intensity. b . The dose response of one Opto-CheY V416T motor is shown along with best fit curves for fixed values of θ : θ = 2 (black line), θ = 1 (shallower dashed black line), θ = 3 (steeper dashed black line). c . A histogram of most probable θ for 20 individual BFM response curves, corresponding to the fit with highest R 2 .

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: a . Opto-CheY V416T -driven motor responses. Each motor is flashed with a series of pulses of increasing relative intensity ( I = I min · x, x = {1, 2, 4, 8, 16, 32}, I min delivers ~ 7 · 10 12 blue photons/cm 2 ). Response probability for each BFM at a given intensity is calculated as the number of responses divided by the number of flashes. P see with θ = 2 is used to fit each dose response curve . Ten individual BFM response curves and fits are shown translated horizontally relative to half-maximum response intensity. b . The dose response of one Opto-CheY V416T motor is shown along with best fit curves for fixed values of θ : θ = 2 (black line), θ = 1 (shallower dashed black line), θ = 3 (steeper dashed black line). c . A histogram of most probable θ for 20 individual BFM response curves, corresponding to the fit with highest R 2 .

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques:

Opto-CheY V416T , Opto-CheY N449S , and Opto-Che single motor dose response curves, as indicated. Each motor responds to light pulses of increasing relative intensity ( I = I min · x, x = {1, 2, 4, 8, 16, 32}) controlled using neutral density filters. Response probability for each BFM expressing an Opto-CheY variant at a given blue light intensity is calculated as the number of responses divided by the number of flashes. P see with θ = 2 fits are shown for each of the 20 Opto-CheY V416T dose response measurements in corresponding color. R 2 values for the fits are given in . For Opto-CheY N449S strains, data is fitted to b× P see , where b is between 0 and 1, a free parameter to allow response probability saturation at values less than 1.

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: Opto-CheY V416T , Opto-CheY N449S , and Opto-Che single motor dose response curves, as indicated. Each motor responds to light pulses of increasing relative intensity ( I = I min · x, x = {1, 2, 4, 8, 16, 32}) controlled using neutral density filters. Response probability for each BFM expressing an Opto-CheY variant at a given blue light intensity is calculated as the number of responses divided by the number of flashes. P see with θ = 2 fits are shown for each of the 20 Opto-CheY V416T dose response measurements in corresponding color. R 2 values for the fits are given in . For Opto-CheY N449S strains, data is fitted to b× P see , where b is between 0 and 1, a free parameter to allow response probability saturation at values less than 1.

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Expressing, Variant Assay

Averaged binary traces (100 frames running average, 500 msec) are shown for strain 630 . Tethered cells (Opto-CheY N449S ) are subjected to 2 msec pulses every 6 sec at the highest intensity (vertical blue lines). Following are 20 sec pulses (horizontal blue lines) at 1 / 32 of the maximum intensity.

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: Averaged binary traces (100 frames running average, 500 msec) are shown for strain 630 . Tethered cells (Opto-CheY N449S ) are subjected to 2 msec pulses every 6 sec at the highest intensity (vertical blue lines). Following are 20 sec pulses (horizontal blue lines) at 1 / 32 of the maximum intensity.

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques:

AlphaFold first ranked model predictions are shown for Opto-CheY N449S and Opto-CheY V416T , color scheme as in . Red arrow heads point to the location of the introduced mutations. Below, the cryo-EM structure of the flagellar hook from Salmonella is shown (pdb ID 7CGB). One FlgE subunit of the hook is highlighted in blue. AlphaFold structure prediction of FlgE AviRRR is shown to the right (engineered loop in red).

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: AlphaFold first ranked model predictions are shown for Opto-CheY N449S and Opto-CheY V416T , color scheme as in . Red arrow heads point to the location of the introduced mutations. Below, the cryo-EM structure of the flagellar hook from Salmonella is shown (pdb ID 7CGB). One FlgE subunit of the hook is highlighted in blue. AlphaFold structure prediction of FlgE AviRRR is shown to the right (engineered loop in red).

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Cryo-EM Sample Prep

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