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Novus Biologicals nb100 1770ss rrid ab 10128178
Nb100 1770ss Rrid Ab 10128178, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 96/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+gfp/GFP+Antibody/pmc13651822-2-6-3
Average 96 stars, based on 133 article reviews

nb100 1770ss rrid ab 10128178 - by Bioz Stars, 2026-10

96/100 stars

Images

Cell-Signaling:

Article Title: Intrinsic resistance to RAS inhibitors is driven by dysregulation of KRAS degradation
Article Snippet: Mouse monoclonal , Anti-Vinculin (Sigma Aldrich, clone hVIN-1, V 9131), anti KRAS-G12D mutant (GTX635362, GeneTex), Anti-RIT1 (GTX117329, GeneTex), Anti-Actin α-Smooth Muscle - Cy3 (Sigma Aldrich, C6198), anti CD107a (LAMP-1) (Biolegend, 328601), Anti-4EBP1 Phospho (Thr70) (Biolegend, 619601), Anti-mTOR (Biolegend, 659202), anti CD98 (SLC3A2) (Biolegend, 315602), Anti-Actin, α-Smooth Muscle - Cy3 antibody (Sigma-Aldrich, C6198), Anti-Flag M2 Antibody (Sigma-Aldrich, F3165).. .. Rabbit , Anti-Cytokeratin 19 (10712-1-AP, Proteintech), Anti-FLAG Epitope (DYKDDDDK) (740001, ThermoFisher), Anti-GATA6 (55435-1-AP, Proteintech), Anti-GFP (NB600-308, Novus Biologicals), anti HIF1a (H1alpha67, Novus Biologicals), Anti-KRAS (12063-1-AP, Proteintech), anti TTF1 (66034-1-Ig, Proteintech), Anti-Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signaling, 9234S), Anti-p70 S6 Kinase (108D2) (Cell Signaling, 9202S), Anti-4EBP1 (Sanbio, 60246), Anti-mTOR (7C10) (Cell Signaling, #2983), LAT1/SLC7A5 (E9O4D) (Cell Signaling, 32683S), Anti-4F2hc/SLC3A2 (D6O3P) (Cell Signaling, 13180S), anti-ubiquitin (ab7780, Abcam). .. Mouse minus , SLC3A2 (Biolegend, 315602) LAMP1 (Biolegend, 121601).

Western Blot:

Article Title: Focal DEPDC5 loss without disruption to cerebral cortical neuron migration recapitulates DEPDC5-related focal epilepsy.
Article Snippet: Gels were transferred onto Immobilon-FL PVDF membranes (Millipore), blocked with Odyssey TBS blocking buffer (LI-COR) for 1 hour at room temperature, followed by primary antibody in Odyssey blocking buffer with 0.2% Tween 20 (Sigma-Aldrich) overnight at 4°C. .. Antibodies used for Western blotting were as follows: anti–β-actin (Cell Signaling Technology, catalog 3700; 1:5000), anti–p-S6 (Ser 240/Ser244) (Cell Signaling Technology, catalog 5364; 1:1000), anti-S6 (Santa Cruz Biotechnology, catalog sc-74459; 1:1000), anti–total Akt (Cell Signaling Technology, catalog 4691; 1:1000), anti–p-Akt Ser473 (Cell Signaling Technology, catalog 4060, 1:1000), anti-DEPDC5 (Sigma-Aldrich, catalog HPA055619; 1:1000), anti-GFP (Novus Biological, catalog NB600-308; 1:1000), and antimTOR (Cell Signaling Technology, catalog 2983S; 1:1000). .. After washing blots 3 times for 5 minutes each in Tris-buffered saline–Tween 20 (TBST), IRDye 800CW– or 680RD–conjugated secondary antibodies (1:10,000; LI-COR) in Odyssey blocking buffer with 0.2% Tween 20 and and 0.01% SDS were added for 1 hour at room temperature.

RNAscope:

Article Title: Conserved cellular architecture and developmental mechanisms of the zebrafish meninges
Article Snippet: .. RNAscope experiments were followed by immunofluorescence or performed independently using the same antigen retrieval reagents as the RNAscope Fluorescent Multiplex kit, and slides were blocked for 1 h in 10% (adult stains) or 5% (embryonic stains) goat serum, stained with primary overnight (anti-laminin, 1:250, Millipore #L9393; anti-GFP, 1:1000, Novus NB100-1614; anti-mCherry, 1:1000, Novus NBP2-25157) and stained with Alexa-Fluor secondaries from Thermo Scientific (1:250, Thermo) and Hoechst 33342 at room temperature for 1 h. Wholemount staining of adult brains were performed by permeabilizing tissues with 0.25% Triton for 3 hours at room temperature followed by blocking in anti-acetylated α-tubulin antibody (1:500, Sigma T6793) overnight, and the secondary for two hours at room temperature with secondary antibody and Hoechst 33342. .. RNAscope reagents were purchased from Advanced Cell Diagnostics, and experiments were performed using the RNAscope Fluorescent Multiplex V2 Assay according to the manufacturer’s protocol for formalin-fixed paraffin-embedded (adult) or for fixed frozen (embryos) sections.

Immunofluorescence:

Multiplex Assay:

Staining:

Blocking Assay:

other:

Article Title: Drosophila Keap1 Proteins Assemble Nuclear Condensates in Response to Oxidative Stress.
Article Snippet: Samples were imaged, and movies were captured using a Nikon Eclipse Ni fluorescence microscope (Nikon USA, Melville, NY, USA) with 20× and 40× objectives.

Article Title: Intrinsic resistance to RAS inhibitors is driven by dysregulation of KRAS degradation.
Article Snippet: The following antibodies/dyes were used: Mouse monoclonal Anti-Vinculin (Sigma Aldrich, clone hVIN-1, V 9131), anti KRAS-G12D mutant (GTX635362, GeneTex), Anti-RIT1 (GTX117329, GeneTex), AntiActin α-Smooth Muscle - Cy3 (Sigma Aldrich, C6198), anti CD107a (LAMP1) (Biolegend, 328601), Anti-4EBP1 Phospho (Thr70) (Biolegend, 619601), Anti-mTOR (Biolegend, 659202), anti CD98 (SLC3A2) (Biolegend, 315602), AR TI CL E IN P RE SS ARTICLE IN PRESS Anti-Actin, α-Smooth Muscle - Cy3 antibody (Sigma-Aldrich, C6198), AntiFlag M2 Antibody (Sigma-Aldrich, F3165).

Article Title: Drosophila Keap1 Proteins Assemble Nuclear Condensates in Response to Oxidative Stress
Article Snippet: Samples were imaged, and movies were captured using a Nikon Eclipse Ni fluorescence microscope (Nikon USA, Melville, NY, USA) with 20× and 40× objectives.

Immunostaining:

Article Title: TP53/TAU axis regulates microtubule bundling to control alveolar stem cell–mediated regeneration
Article Snippet: Images were recorded using a 20× objective of the Axio imager (ZEISS). .. The following antibodies and dyes were applied to samples for immunostaining: anti-RAGE/AGER (MAB1179, R&D Systems, 1:500), anti-proSP-C (AB3786, MilliporeSigma, 1:500), anti-ABCA3 (3C9) (sc58220, Santa Cruz Biotechnology, 1:300), anti-Claudin4 (36-4800, Invitrogen, 1:200), anti-Actin, alpha-Smooth Muscle Cy3-conjugated (C6198, MilliporeSigma,1:500), anti-HT1-56 (TB-29AHT1-56, Terrace Biotech, 1:300), anti-HTII-280 (TB-27AHT2-280, Terrace Biotech, 1:50), anti-GFP (NB100-1770, Novus Biologicals, 1:500), anti-tdTomato (AB8181-200, OriGene, 1:1,000), anti-TUBA1A antibody (PA5-22060, Invitrogen, 1:100), anti-TUBA1B (66031-1-Ig, Proteintech, 1:500), anti-acetylated Tubulin (66200-1-Ig, Proteintech, 1:500), anti-Tau (10274-1-AP, Proteintech, 1:200), anti-Tau-1 (PC1C6) (MAB3420, MilliporeSigma,1:100), anti-Tau (Tau-5) (AHB0042, Invitrogen,1:50), anti-MAP2 (17490-1-AP, Proteintech, 1:500), anti-Keratin8 (TROMA-I, DSHB, 1:50), anti-GM130 (610822, BD Biosciences, 1:50), Alexa Fluor-555 Phalloidin (A34055, Invitrogen, 1:400), Alexa Fluor-647 Phalloidin (A22287, Invitrogen, 1:400), LEL-DyLight-649 (DL-1178, Vector Laboratories,1:1,500), anti-FLAG-M2 (F1804, Sigma-Aldrich, 1;1,000), anti-Calnexin (AB22595, Abcam, 1:500), anti-CD107a/LAMP-1 (121601, BioLegend, 1:500), anti-SFN (PA5-95056, Invitrogen, 1:250), anti-LGALS3-Alexa647 (125408, BioLegend, 1:500), and anti-TAGLN/Transgelin (ab14106, Abcam, 1:250). .. Images were captured using an Olympus FV3000 confocal microscope with 20×, 30×, 40×, and 60× objectives.

Image Search Results

The BmMKLP1 domains exhibit divergent nuclear and cytoplasmic localization (A) Subcellular localization of transiently expressed GFP-tagged domains (green) in BmN4 cells. Representative merged bright-field and GFP fluorescence images acquired at 10×, scale bars, 100 μm, BmN4 cells expressing the indicated constructs were imaged by differential interference contrast (DIC) microscopy (left) to show cell morphology and by fluorescence microscopy (right) to detect GFP-tagged proteins. All images were acquired with a 40× objective lens. Scale bars, 20 μm. (B) Western blot analysis of protein extracts from transfected BmN4 cells probed with an anti-GFP antibody (1:1,000, Proteintech, Wuhan, China), confirming the production of each GFP-tagged domain at its expected molecular weight. The “control” lane corresponds to the cycle 3 GFP-bleo fusion protein (∼39–40 KDa) expressed from the pIZT/V5-His vector. A representative blot from three independent experiments is shown. The PageRuler Prestained Protein Ladder (10–180 kDa; Thermo Fisher Scientific, Waltham, MA, USA) was used as a molecular weight marker.

Journal: iScience

Article Title: Domain-autonomous MKLP1 modules divergently regulate mitotic fidelity and polyploidy in a holocentric system

doi: 10.1016/j.isci.2026.117149

Figure Lengend Snippet: The BmMKLP1 domains exhibit divergent nuclear and cytoplasmic localization (A) Subcellular localization of transiently expressed GFP-tagged domains (green) in BmN4 cells. Representative merged bright-field and GFP fluorescence images acquired at 10×, scale bars, 100 μm, BmN4 cells expressing the indicated constructs were imaged by differential interference contrast (DIC) microscopy (left) to show cell morphology and by fluorescence microscopy (right) to detect GFP-tagged proteins. All images were acquired with a 40× objective lens. Scale bars, 20 μm. (B) Western blot analysis of protein extracts from transfected BmN4 cells probed with an anti-GFP antibody (1:1,000, Proteintech, Wuhan, China), confirming the production of each GFP-tagged domain at its expected molecular weight. The “control” lane corresponds to the cycle 3 GFP-bleo fusion protein (∼39–40 KDa) expressed from the pIZT/V5-His vector. A representative blot from three independent experiments is shown. The PageRuler Prestained Protein Ladder (10–180 kDa; Thermo Fisher Scientific, Waltham, MA, USA) was used as a molecular weight marker.

Article Snippet: Scale bars, 20 μm. (B) Western blot analysis of protein extracts from transfected BmN4 cells probed with an anti-GFP antibody (1:1,000, Proteintech, Wuhan, China), confirming the production of each GFP-tagged domain at its expected molecular weight.

Techniques: Fluorescence, Expressing, Construct, Microscopy, Morphology, Western Blot, Analysis, Transfection, Molecular Weight, Control, Plasmid Preparation, Marker

The head and stalk domains induce G2/M arrest, whereas the tail promotes polyploidization (A) Representative flow cytometry histograms showing DNA content profiles (top) and polyploidy distributions (bottom) for control cells and cells expressing GFP-tagged BmMKLP1—full-length, head, -stalk, or -tail. Cell cycle phases (G0/G1, S, and G2/M) were determined by model-based fitting of propidium iodide (PI) fluorescence intensity histograms. Polyploidy histograms depict cells with DNA content exceeding 4 N. (B) Quantification of cell cycle distribution under each condition. Bars represent the mean ± SEM percentage of cells in G0/G1, S, and G2/M phases from at least three independent experiments. Different lowercase letters indicate significant differences (one-way ANOVA followed by Tukey’s HSD, p < 0.05). (C) Percentage of polyploid cells under each condition. Bars represent mean ± SEM, with statistical significance assessed as in (B).

Journal: iScience

Article Title: Domain-autonomous MKLP1 modules divergently regulate mitotic fidelity and polyploidy in a holocentric system

doi: 10.1016/j.isci.2026.117149

Figure Lengend Snippet: The head and stalk domains induce G2/M arrest, whereas the tail promotes polyploidization (A) Representative flow cytometry histograms showing DNA content profiles (top) and polyploidy distributions (bottom) for control cells and cells expressing GFP-tagged BmMKLP1—full-length, head, -stalk, or -tail. Cell cycle phases (G0/G1, S, and G2/M) were determined by model-based fitting of propidium iodide (PI) fluorescence intensity histograms. Polyploidy histograms depict cells with DNA content exceeding 4 N. (B) Quantification of cell cycle distribution under each condition. Bars represent the mean ± SEM percentage of cells in G0/G1, S, and G2/M phases from at least three independent experiments. Different lowercase letters indicate significant differences (one-way ANOVA followed by Tukey’s HSD, p < 0.05). (C) Percentage of polyploid cells under each condition. Bars represent mean ± SEM, with statistical significance assessed as in (B).

Techniques: Flow Cytometry, Control, Expressing, Fluorescence

Journal: iScience

Article Title: Domain-autonomous MKLP1 modules divergently regulate mitotic fidelity and polyploidy in a holocentric system

doi: 10.1016/j.isci.2026.117149

Figure Lengend Snippet:

Techniques: Transfection, Cloning, Imaging, Bicinchoninic Acid Protein Assay, Western Blot, Plasmid Preparation, Recombinant, Software

Journal: Cell

Article Title: The structural basis for LRRK2’s activation and autoinhibition

doi: 10.1016/j.cell.2026.07.027

Figure Lengend Snippet:

Article Snippet: GFP , Santa Cruz Biotechnology , Cat# sc-9996; Lot# C2523; RRID:AB_627695.

Techniques: Cell-Signaling, Virus, Recombinant, Plasmid Preparation, Software, Protease Inhibitor

(A) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of NPR1 phosphorylation. 35S:NPR1-GFP plants were treated with mock or 0.5 mM SA for 24 hours, followed by trypsin (top)/Glu-C (bottom) digestion for LC-MS/MS. (B) Phosphorylation of NPR1 at Ser-557. Top: 3.5-week-old 35S:NPR1-GFP plants were treated with mock, 0.1 or 1 mM SA 24 hours before collection, or inoculated with Psm avrRpt2 ( avrRpt2 , OD 600 = 0.01) in local leaves 2 days before systemic collection. Bottom: 3.5-week-old wild-type (WT) plants were treated with mock or 1 mM SA 8 hours before collection. NPR1-GFP and endogenous NPR1 were immunoprecipitated using GFP-trap and α-NPR1, respectively, and then blotted with an anti-phosphorylated Ser-557 (α-pS557) antibody. Relative phosphorylation levels (top) were calculated by normalizing band intensities to total NPR1-GFP levels. (C and D) Representative image (C) and NPR1 protein levels (D) of 3.5-week-old WT, npr1-2 , 35S:NPR1-GFP , and independent 35S:npr1 S557A - GFP and 35S:npr1 S557D - GFP transgenic lines. Scale bar, 5 cm. (E) Basal resistance in 35S:NPR1-GFP , 35S:npr1 S557A - GFP, 35S:npr1 S557D - GFP , and npr1-2 plants inoculated with Psm (OD 600 = 0.0002). Bacterial growth was measured at 3 days post-inoculation (dpi) in all experiments. cfu, colony-forming units. n = 8. (F) SA-induced resistance in 35S:NPR1-GFP , 35S:npr1 S557A - GFP , 35S:npr1 S557D - GFP , and npr1-2 plants. 3.5-week-old plants were treated with mock or 1 mM SA, followed by inoculation with Psm (OD 600 = 0.001) 24 hours later. n = 8. (G) PR1 gene expression. RT-qPCR was performed in 3.5-week-old 35S:NPR1-GFP , 35S:npr1 S557A - GFP , and 35S:npr1 S557D - GFP plants 24 hours after mock, 0.1 mM, or 1 mM SA treatment. UBQ5 expression was used for normalization in all RT-qPCR experiments. (H and I) Representative image (H) of and NPR1 protein levels (I) of 3.5-week-old WT, npr1-2 , and independent NPR1pro:NPR1-eYFP , NPR1pro:npr1 S557A - eYFP , and NPR1pro:npr1 S557D - eYFP transgenic lines. Scale bar, 5 cm. Samples in (I) were collected 16 hours after mock or 0.1 mM SA treatment. (J) Basal and SA-induced resistance in WT, npr1-2 , NPR1pro:NPR1-eYFP ( NPR1 ), NPR1pro:npr1 S557A - eYFP ( npr1 S557A ), and NPR1pro:npr1 S557D - eYFP ( npr1 S557D ) plants treated with mock or 1 mM SA 24 hours before inoculation with Psm (OD 600 = 0.001). n = 8. (K) PR1 gene expression. RT-qPCR was performed in 3.5-week-old WT, npr1-2 , NPR1pro:NPR1-eYFP ( NPR1 ), NPR1pro:npr1 S557A - eYFP ( npr1 S557A ), and NPR1pro:npr1 S557D - eYFP ( npr1 S557D ) plants 24 hours after mock, 0.1 mM SA, or 1 mM SA treatment. All data in the manuscript are presented as mean ± s.e.m. Individual columns were compared using one-way ANOVA with Tukey’s post hoc (E, F, and J) or two-tailed Student’s t-test (G and K), and the interactions were tested using two-way ANOVA (F and J). For all figures, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant; different lowercase letters indicate significant differences between groups by Tukey’s post hoc at p < 0.05. See also Figure S1 and Table S1 .

Journal: Cell

Article Title: Salicylic Acid Engages Central Metabolic Regulators SnRK1 and TOR to Govern Immunity by Differential Phosphorylation of NPR1

doi: 10.1016/j.cell.2026.09.011

Figure Lengend Snippet: (A) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of NPR1 phosphorylation. 35S:NPR1-GFP plants were treated with mock or 0.5 mM SA for 24 hours, followed by trypsin (top)/Glu-C (bottom) digestion for LC-MS/MS. (B) Phosphorylation of NPR1 at Ser-557. Top: 3.5-week-old 35S:NPR1-GFP plants were treated with mock, 0.1 or 1 mM SA 24 hours before collection, or inoculated with Psm avrRpt2 ( avrRpt2 , OD 600 = 0.01) in local leaves 2 days before systemic collection. Bottom: 3.5-week-old wild-type (WT) plants were treated with mock or 1 mM SA 8 hours before collection. NPR1-GFP and endogenous NPR1 were immunoprecipitated using GFP-trap and α-NPR1, respectively, and then blotted with an anti-phosphorylated Ser-557 (α-pS557) antibody. Relative phosphorylation levels (top) were calculated by normalizing band intensities to total NPR1-GFP levels. (C and D) Representative image (C) and NPR1 protein levels (D) of 3.5-week-old WT, npr1-2 , 35S:NPR1-GFP , and independent 35S:npr1 S557A - GFP and 35S:npr1 S557D - GFP transgenic lines. Scale bar, 5 cm. (E) Basal resistance in 35S:NPR1-GFP , 35S:npr1 S557A - GFP, 35S:npr1 S557D - GFP , and npr1-2 plants inoculated with Psm (OD 600 = 0.0002). Bacterial growth was measured at 3 days post-inoculation (dpi) in all experiments. cfu, colony-forming units. n = 8. (F) SA-induced resistance in 35S:NPR1-GFP , 35S:npr1 S557A - GFP , 35S:npr1 S557D - GFP , and npr1-2 plants. 3.5-week-old plants were treated with mock or 1 mM SA, followed by inoculation with Psm (OD 600 = 0.001) 24 hours later. n = 8. (G) PR1 gene expression. RT-qPCR was performed in 3.5-week-old 35S:NPR1-GFP , 35S:npr1 S557A - GFP , and 35S:npr1 S557D - GFP plants 24 hours after mock, 0.1 mM, or 1 mM SA treatment. UBQ5 expression was used for normalization in all RT-qPCR experiments. (H and I) Representative image (H) of and NPR1 protein levels (I) of 3.5-week-old WT, npr1-2 , and independent NPR1pro:NPR1-eYFP , NPR1pro:npr1 S557A - eYFP , and NPR1pro:npr1 S557D - eYFP transgenic lines. Scale bar, 5 cm. Samples in (I) were collected 16 hours after mock or 0.1 mM SA treatment. (J) Basal and SA-induced resistance in WT, npr1-2 , NPR1pro:NPR1-eYFP ( NPR1 ), NPR1pro:npr1 S557A - eYFP ( npr1 S557A ), and NPR1pro:npr1 S557D - eYFP ( npr1 S557D ) plants treated with mock or 1 mM SA 24 hours before inoculation with Psm (OD 600 = 0.001). n = 8. (K) PR1 gene expression. RT-qPCR was performed in 3.5-week-old WT, npr1-2 , NPR1pro:NPR1-eYFP ( NPR1 ), NPR1pro:npr1 S557A - eYFP ( npr1 S557A ), and NPR1pro:npr1 S557D - eYFP ( npr1 S557D ) plants 24 hours after mock, 0.1 mM SA, or 1 mM SA treatment. All data in the manuscript are presented as mean ± s.e.m. Individual columns were compared using one-way ANOVA with Tukey’s post hoc (E, F, and J) or two-tailed Student’s t-test (G and K), and the interactions were tested using two-way ANOVA (F and J). For all figures, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant; different lowercase letters indicate significant differences between groups by Tukey’s post hoc at p < 0.05. See also Figure S1 and Table S1 .

Article Snippet: NPR1-GFP was purified from the extracts using GFP-Trap agarose beads (Chromotek) following the manufacturer’s instructions.

Techniques: Phospho-proteomics, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Analysis, Immunoprecipitation, Transgenic Assay, Gene Expression, Quantitative RT-PCR, Expressing, Two Tailed Test

$(A) Subcellular localization of NPR1 Ser-557 phosphorylation. Seven-day-old 35S:NPR1-GFP seedlings were treated with mock or 0.3 mM SA for 6 hours. After subcellular fractionation, nuclear and cytoplasmic proteins were blotted with α-pS557. Total NPR1 levels were detected using α-GFP, and fraction purity was determined using α-UGPase and α-Histone H3. *, nonspecific band. (B and C) Time course of SA-induced NPR1 phosphorylation. 35S:NPR1-GFP and npr1-2 seedlings were treated with 0.3 mM SA for the indicated durations. NPR1 phosphorylation was detected using α-pS55/59, α-pS11/15, and α-pS557 (B) and quantified by normalizing to the total NPR1 levels (α-GFP) (C). For each blot, the band with the highest relative phosphorylation level was set to 1. n = 3. (D) Hierarchy in NPR1 PTMs. 35S:NPR1-GFP , 35S:npr1 S55/59A - GFP , 35S:npr1 S55/59D - GFP , 35S:npr1 sim3 - GFP , and npr1-2 seedlings were treated with mock or 0.3 mM SA for 6 hours. Proteins were analyzed using α-pS557, α-pS11/15, and α-GFP. (E and F) Analysis of Ser-55/59 and Ser-11/15 phosphorylation in Ser-557 phospho-mutant lines. 35S:NPR1-GFP , independent 35S:npr1 S557A - GFP , and 35S:npr1 S557D - GFP transgenic lines, and npr1-2 seedlings were treated as in (D). Proteins were analyzed with α-pS55/59 (E), α-pS11/15 (F), and α-GFP. (G and H) Quantification of Ser-11/15 phosphorylation in Ser-557 mutants from four independent experiments. SA-induced fold changes in Ser-11/15 phosphorylation of NPR1-GFP, npr1 S557A -GFP, and npr1 S557D -GFP relative to mock were calculated after normalizing each band intensity to its loading control (G). SA-induced Ser-11/15 phosphorylation in npr1 S557D -GFP relative to that observed in NPR1-GFP was calculated (H). n = 4. (I) Analysis of Ser-557 phosphorylation in Ser-11/15 phospho-mutant lines. 35S:NPR1-GFP , 35S:npr1 S11/15A - GFP , 35S:npr1 S11/15D - GFP , and npr1-2 seedlings were treated and analyzed as in (D) using α-pS557 and α-GFP. (J) Analysis of NPR1 ubiquitination in Ser-557 phospho-mutant lines. 35S:NPR1-GFP , 35S:npr1 S557A - GFP , and 35S:npr1 S557D - GFP seedlings were treated as in (D). Ubiquitinated proteins were immunoprecipitated using Ubiquitin-trap. NPR1 was detected using α-GFP. The intensity of the upper, ubiquitinated fraction (>90 kDa) was normalized to total NPR1 levels. Individual columns were compared using one-way ANOVA with Tukey’s post hoc test (C) or two-tailed Student’s t-test (G and H). Total protein loading for (B, D-F, and I) was estimated using Ponceau S staining. See also Figure S1 .$

Journal: Cell

Article Title: Salicylic Acid Engages Central Metabolic Regulators SnRK1 and TOR to Govern Immunity by Differential Phosphorylation of NPR1

doi: 10.1016/j.cell.2026.09.011

Figure Lengend Snippet: (A) Subcellular localization of NPR1 Ser-557 phosphorylation. Seven-day-old 35S:NPR1-GFP seedlings were treated with mock or 0.3 mM SA for 6 hours. After subcellular fractionation, nuclear and cytoplasmic proteins were blotted with α-pS557. Total NPR1 levels were detected using α-GFP, and fraction purity was determined using α-UGPase and α-Histone H3. *, nonspecific band. (B and C) Time course of SA-induced NPR1 phosphorylation. 35S:NPR1-GFP and npr1-2 seedlings were treated with 0.3 mM SA for the indicated durations. NPR1 phosphorylation was detected using α-pS55/59, α-pS11/15, and α-pS557 (B) and quantified by normalizing to the total NPR1 levels (α-GFP) (C). For each blot, the band with the highest relative phosphorylation level was set to 1. n = 3. (D) Hierarchy in NPR1 PTMs. 35S:NPR1-GFP , 35S:npr1 S55/59A - GFP , 35S:npr1 S55/59D - GFP , 35S:npr1 sim3 - GFP , and npr1-2 seedlings were treated with mock or 0.3 mM SA for 6 hours. Proteins were analyzed using α-pS557, α-pS11/15, and α-GFP. (E and F) Analysis of Ser-55/59 and Ser-11/15 phosphorylation in Ser-557 phospho-mutant lines. 35S:NPR1-GFP , independent 35S:npr1 S557A - GFP , and 35S:npr1 S557D - GFP transgenic lines, and npr1-2 seedlings were treated as in (D). Proteins were analyzed with α-pS55/59 (E), α-pS11/15 (F), and α-GFP. (G and H) Quantification of Ser-11/15 phosphorylation in Ser-557 mutants from four independent experiments. SA-induced fold changes in Ser-11/15 phosphorylation of NPR1-GFP, npr1 S557A -GFP, and npr1 S557D -GFP relative to mock were calculated after normalizing each band intensity to its loading control (G). SA-induced Ser-11/15 phosphorylation in npr1 S557D -GFP relative to that observed in NPR1-GFP was calculated (H). n = 4. (I) Analysis of Ser-557 phosphorylation in Ser-11/15 phospho-mutant lines. 35S:NPR1-GFP , 35S:npr1 S11/15A - GFP , 35S:npr1 S11/15D - GFP , and npr1-2 seedlings were treated and analyzed as in (D) using α-pS557 and α-GFP. (J) Analysis of NPR1 ubiquitination in Ser-557 phospho-mutant lines. 35S:NPR1-GFP , 35S:npr1 S557A - GFP , and 35S:npr1 S557D - GFP seedlings were treated as in (D). Ubiquitinated proteins were immunoprecipitated using Ubiquitin-trap. NPR1 was detected using α-GFP. The intensity of the upper, ubiquitinated fraction (>90 kDa) was normalized to total NPR1 levels. Individual columns were compared using one-way ANOVA with Tukey’s post hoc test (C) or two-tailed Student’s t-test (G and H). Total protein loading for (B, D-F, and I) was estimated using Ponceau S staining. See also Figure S1 .

Article Snippet: NPR1-GFP was purified from the extracts using GFP-Trap agarose beads (Chromotek) following the manufacturer’s instructions.

Techniques: Phospho-proteomics, Gene Expression, Cell Fractionation, Analysis, Mutagenesis, Transgenic Assay, Control, Ubiquitin Proteomics, Immunoprecipitation, Two Tailed Test, Staining

(A) In vitro phosphorylation assays of recombinant NPR1. Ser-557 and total phosphorylation levels were detected using α-pS557 and α-pS/pT, respectively. In vitro SnRK1 activity was estimated by measuring phosphorylation of its T-loop using α-pAMPK, and total SnRK1/SnAK1 proteins were detected using α-FLAG. Total NPR1 was stained using Coomassie Brilliant Blue (CBB). *, nonspecific band. (B) LC-MS/MS analysis of in vitro phosphorylated GST-NPR1 by SnRK1. Extracted ion chromatograms (XICs) of MS2 fragment ions over retention time for the most confidently detected peptide (AFS[Phos]EDNLELGNSSLTDSTSSTSK) are shown for each group. (C) Interaction of GFP-fused SnRK1α with NPR1-mCherry or HA-mCherry transiently expressed in N. benthamiana, evaluated 6 hours after 1 mM SA treatment. (D) Interaction between endogenous SnRK1α and NPR1-GFP or GFP in transgenic Arabidopsis seedlings treated with water or 0.3 mM SA for 6 hours. SnRK1α intensities for the mock samples were set to 1 for normalization. (E) In vitro interactions between SnRK1α, SnAK1, and GST-fused NPR1. (F) Effect of snrk1 mutations on Ser-557 phosphorylation. NPR1-GFP was transiently expressed in WT and snrk1α1 −/− snrk1α2 +/− ( sesquiα2-1 ) leaf-derived protoplasts, followed by treatment with mock or 0.1 mM SA for 6 hours. NPR1-GFP was immunoprecipitated and analyzed with α-pS557 and α-GFP. Relative phosphorylation levels were calculated after normalization to total NPR1-GFP, with the mock-treated WT sample set to 1. (G) Effect of snrk1 mutations on Ser-557 phosphorylation of endogenous NPR1. 3.5-week-old WT and sesquiα2-1 plants were treated with mock or 1 mM SA 8 hours before collection. NPR1 protein was immunoprecipitated using α-NPR1 and analyzed using α-pS557 and α-NPR1. Relative phosphorylation levels were calculated as in (F). *, IgG heavy chain band from IP. (H) Effect of snrk1 mutations on endogenous NPR1 Ser-557 phosphorylation during biological SAR induction. 3.5-week-old WT and sesquiα2-1 plants were inoculated with MgCl 2 or Psm avrRpt2 (OD 600 = 0.01) in two local leaves, followed by collection of three systemic leaves after 2 days. NPR1 was enriched by α-NPR1 IP and analyzed using α-pS557 and α-NPR1. Relative phosphorylation levels were calculated as in (F). *, IgG heavy chain band from IP. (I) Effect of snrk1 mutations on Ser-557 phosphorylation in stable transgenic lines. NPR1-eYFP seedlings in npr1-7 ( npr1 ) or npr1 sesquiα2-1 ( npr1 α2–1 ) backgrounds were treated with mock or 0.3 mM SA for 6 hours. Proteins were analyzed using α-pS557 and α-GFP. Relative phosphorylation levels were calculated as in (F). *, nonspecific band. (J) Requirement of SA biosynthesis for Ser-557 phosphorylation in systemic tissue. 3.5-week-old WT and SA-deficient sid2-2 plants were inoculated with Psm avrRpt2 (OD 600 = 0.01) in local leaves, followed by collection of systemic leaves at the indicated time points. NPR1 protein was enriched through α-NPR1 IP and analyzed using α-pS557 and α-NPR1. Relative phosphorylation levels were calculated by normalizing to the corresponding total protein levels, with the WT sample at 12 hpi set to 1. See also Figure S2 .

Journal: Cell

Article Title: Salicylic Acid Engages Central Metabolic Regulators SnRK1 and TOR to Govern Immunity by Differential Phosphorylation of NPR1

doi: 10.1016/j.cell.2026.09.011

Figure Lengend Snippet: (A) In vitro phosphorylation assays of recombinant NPR1. Ser-557 and total phosphorylation levels were detected using α-pS557 and α-pS/pT, respectively. In vitro SnRK1 activity was estimated by measuring phosphorylation of its T-loop using α-pAMPK, and total SnRK1/SnAK1 proteins were detected using α-FLAG. Total NPR1 was stained using Coomassie Brilliant Blue (CBB). *, nonspecific band. (B) LC-MS/MS analysis of in vitro phosphorylated GST-NPR1 by SnRK1. Extracted ion chromatograms (XICs) of MS2 fragment ions over retention time for the most confidently detected peptide (AFS[Phos]EDNLELGNSSLTDSTSSTSK) are shown for each group. (C) Interaction of GFP-fused SnRK1α with NPR1-mCherry or HA-mCherry transiently expressed in N. benthamiana, evaluated 6 hours after 1 mM SA treatment. (D) Interaction between endogenous SnRK1α and NPR1-GFP or GFP in transgenic Arabidopsis seedlings treated with water or 0.3 mM SA for 6 hours. SnRK1α intensities for the mock samples were set to 1 for normalization. (E) In vitro interactions between SnRK1α, SnAK1, and GST-fused NPR1. (F) Effect of snrk1 mutations on Ser-557 phosphorylation. NPR1-GFP was transiently expressed in WT and snrk1α1 −/− snrk1α2 +/− ( sesquiα2-1 ) leaf-derived protoplasts, followed by treatment with mock or 0.1 mM SA for 6 hours. NPR1-GFP was immunoprecipitated and analyzed with α-pS557 and α-GFP. Relative phosphorylation levels were calculated after normalization to total NPR1-GFP, with the mock-treated WT sample set to 1. (G) Effect of snrk1 mutations on Ser-557 phosphorylation of endogenous NPR1. 3.5-week-old WT and sesquiα2-1 plants were treated with mock or 1 mM SA 8 hours before collection. NPR1 protein was immunoprecipitated using α-NPR1 and analyzed using α-pS557 and α-NPR1. Relative phosphorylation levels were calculated as in (F). *, IgG heavy chain band from IP. (H) Effect of snrk1 mutations on endogenous NPR1 Ser-557 phosphorylation during biological SAR induction. 3.5-week-old WT and sesquiα2-1 plants were inoculated with MgCl 2 or Psm avrRpt2 (OD 600 = 0.01) in two local leaves, followed by collection of three systemic leaves after 2 days. NPR1 was enriched by α-NPR1 IP and analyzed using α-pS557 and α-NPR1. Relative phosphorylation levels were calculated as in (F). *, IgG heavy chain band from IP. (I) Effect of snrk1 mutations on Ser-557 phosphorylation in stable transgenic lines. NPR1-eYFP seedlings in npr1-7 ( npr1 ) or npr1 sesquiα2-1 ( npr1 α2–1 ) backgrounds were treated with mock or 0.3 mM SA for 6 hours. Proteins were analyzed using α-pS557 and α-GFP. Relative phosphorylation levels were calculated as in (F). *, nonspecific band. (J) Requirement of SA biosynthesis for Ser-557 phosphorylation in systemic tissue. 3.5-week-old WT and SA-deficient sid2-2 plants were inoculated with Psm avrRpt2 (OD 600 = 0.01) in local leaves, followed by collection of systemic leaves at the indicated time points. NPR1 protein was enriched through α-NPR1 IP and analyzed using α-pS557 and α-NPR1. Relative phosphorylation levels were calculated by normalizing to the corresponding total protein levels, with the WT sample at 12 hpi set to 1. See also Figure S2 .

Article Snippet: NPR1-GFP was purified from the extracts using GFP-Trap agarose beads (Chromotek) following the manufacturer’s instructions.

Techniques: In Vitro, Phospho-proteomics, Recombinant, Activity Assay, Staining, Liquid Chromatography with Mass Spectroscopy, Analysis, Transgenic Assay, Derivative Assay, Immunoprecipitation, Tissue

(A-D) Dynamics of SA-induced SnRK1 activation. Seedlings containing the nuclear SnRK1 activity reporter 35S:NLS-ratACC-GFP-HA in WT (A and B) or the snrk1α1 (C and D) background were treated with mock, 0.1 mM SA, or 0.3 mM SA for the indicated durations. Nuclear SnRK1-dependent phosphorylation of the reporter was detected using α-pACC and normalized to total protein levels determined using α-GFP (A and C). The mock-treated sample for each time point was set to 1 (B and D). n = 4. (E) Expression of SnRK1 target genes DIN2 , DIN6 , and SEN5 in response to SA measured by RT-qPCR. 3.5-week-old WT plants were treated with mock, 0.1 mM SA, or 1 mM SA 24 hours before collection. (F) Dynamics of SA-induced expression of SnRK1- and NPR1-target genes DIN2 and PR1 , respectively. 35S:NPR1-GFP seedlings were treated as in Figure 2B , and gene expression was measured by RT-qPCR. (G) Transient SnRK1 overexpression facilitates Ser-557 phosphorylation. Protoplasts derived from leaves of 35S:NPR1-GFP and 35S:npr1 C82A - GFP were transiently transfected with mock, SnRK1α1-mRFP, or the kinase-dead mutant snrk1α1 K48M -mRFP. Proteins were analyzed using α-pS557, α-GFP, and α-mRFP. *, nonspecific band. (H) Effect of darkness-induced SnRK1 activation on Ser-557 phosphorylation. 35S:NPR1-GFP , 35S:npr1 C82A - GFP , and 35S:npr1 C216A - GFP seedlings were kept under light or darkness for 6 hours. Proteins were analyzed using α-pS557 and α-GFP. *, nonspecific band. (I and J) Effects of trehalose 6-phosphate (Tre6P), a SnRK1 inhibitor, on Ser-557 phosphorylation (I) and PR1 expression (J). 35S:NPR1-GFP seedlings grown in sucrose-free ½ MS medium were pretreated with mock or 1 mM Tre6P for 2 hours and subsequently treated with mock or 0.3 mM SA for 6 hours. Relative Ser-557 phosphorylation levels were determined using α-pS557 and α-GFP, whereas PR1 expression levels were determined by RT-qPCR. *, nonspecific band. (K-N) Sugar measurements following SA treatment and biological SAR induction. Levels of Tre6P (K), glucose 6-phosphate (Glc6P; L), trehalose (M), and glucose (N) were measured in 3.5-week-old plants 2 hours and 8 hours after mock or 1 mM SA treatment, or in systemic tissues 24 hours after local SAR induction with Psm avrRpt2 (OD 600 = 0.01) or MgCl 2 . n = 4. (O) Expression of Class I and Class II TPS genes following SA treatment. RT-qPCR was performed in 3.5-week-old WT plants 8 hours after mock or 1 mM SA treatment to measure expression of TPS1 (Class I) as well as TPS5 , TPS6 , and TPS7 (Class II). (P) Expression of all 10 TPP genes following SA treatment. The experiment was performed as in (O). (Q) Expression of TPS1 and TPS5 genes in seedlings was measured after mock or 0.3 mM SA treatment for 2 hours and quantified as in (O). Individual columns were compared using two-tailed Student’s t-test (B, D, E, F, and J-Q) and the interactions were tested using two-way ANOVA (J). Total protein loading for (A, C, H, and I) was estimated using Ponceau S staining. See also Figures S3 and S4 .

Journal: Cell

Article Title: Salicylic Acid Engages Central Metabolic Regulators SnRK1 and TOR to Govern Immunity by Differential Phosphorylation of NPR1

doi: 10.1016/j.cell.2026.09.011

Figure Lengend Snippet: (A-D) Dynamics of SA-induced SnRK1 activation. Seedlings containing the nuclear SnRK1 activity reporter 35S:NLS-ratACC-GFP-HA in WT (A and B) or the snrk1α1 (C and D) background were treated with mock, 0.1 mM SA, or 0.3 mM SA for the indicated durations. Nuclear SnRK1-dependent phosphorylation of the reporter was detected using α-pACC and normalized to total protein levels determined using α-GFP (A and C). The mock-treated sample for each time point was set to 1 (B and D). n = 4. (E) Expression of SnRK1 target genes DIN2 , DIN6 , and SEN5 in response to SA measured by RT-qPCR. 3.5-week-old WT plants were treated with mock, 0.1 mM SA, or 1 mM SA 24 hours before collection. (F) Dynamics of SA-induced expression of SnRK1- and NPR1-target genes DIN2 and PR1 , respectively. 35S:NPR1-GFP seedlings were treated as in Figure 2B , and gene expression was measured by RT-qPCR. (G) Transient SnRK1 overexpression facilitates Ser-557 phosphorylation. Protoplasts derived from leaves of 35S:NPR1-GFP and 35S:npr1 C82A - GFP were transiently transfected with mock, SnRK1α1-mRFP, or the kinase-dead mutant snrk1α1 K48M -mRFP. Proteins were analyzed using α-pS557, α-GFP, and α-mRFP. *, nonspecific band. (H) Effect of darkness-induced SnRK1 activation on Ser-557 phosphorylation. 35S:NPR1-GFP , 35S:npr1 C82A - GFP , and 35S:npr1 C216A - GFP seedlings were kept under light or darkness for 6 hours. Proteins were analyzed using α-pS557 and α-GFP. *, nonspecific band. (I and J) Effects of trehalose 6-phosphate (Tre6P), a SnRK1 inhibitor, on Ser-557 phosphorylation (I) and PR1 expression (J). 35S:NPR1-GFP seedlings grown in sucrose-free ½ MS medium were pretreated with mock or 1 mM Tre6P for 2 hours and subsequently treated with mock or 0.3 mM SA for 6 hours. Relative Ser-557 phosphorylation levels were determined using α-pS557 and α-GFP, whereas PR1 expression levels were determined by RT-qPCR. *, nonspecific band. (K-N) Sugar measurements following SA treatment and biological SAR induction. Levels of Tre6P (K), glucose 6-phosphate (Glc6P; L), trehalose (M), and glucose (N) were measured in 3.5-week-old plants 2 hours and 8 hours after mock or 1 mM SA treatment, or in systemic tissues 24 hours after local SAR induction with Psm avrRpt2 (OD 600 = 0.01) or MgCl 2 . n = 4. (O) Expression of Class I and Class II TPS genes following SA treatment. RT-qPCR was performed in 3.5-week-old WT plants 8 hours after mock or 1 mM SA treatment to measure expression of TPS1 (Class I) as well as TPS5 , TPS6 , and TPS7 (Class II). (P) Expression of all 10 TPP genes following SA treatment. The experiment was performed as in (O). (Q) Expression of TPS1 and TPS5 genes in seedlings was measured after mock or 0.3 mM SA treatment for 2 hours and quantified as in (O). Individual columns were compared using two-tailed Student’s t-test (B, D, E, F, and J-Q) and the interactions were tested using two-way ANOVA (J). Total protein loading for (A, C, H, and I) was estimated using Ponceau S staining. See also Figures S3 and S4 .

Article Snippet: NPR1-GFP was purified from the extracts using GFP-Trap agarose beads (Chromotek) following the manufacturer’s instructions.

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Expressing, Quantitative RT-PCR, Gene Expression, Over Expression, Derivative Assay, Transfection, Mutagenesis, Two Tailed Test, Staining

(A and B) Effect of TOR inhibition on Ser-55/59 phosphorylation. (A) 35S:NPR1-GFP seedlings were treated with mock, 0.3 mM SA (6 hours), the TOR inhibitor Torin 2 (10 μM; 1 or 3 days), or AZD-8055 (2 μM; 1 or 3 days). (B) 35S:NPR1-GFP seedlings were treated with 2 μM AZD-8055 for the indicated durations. Phosphorylated and total NPR1 were detected using α-pS55/59 and α-GFP. (C) Effect of estradiol-induced silencing of TOR on Ser-55/59 phosphorylation. NPR1-GFP was transiently expressed in protoplasts derived from leaves of WT or the estradiol-inducible TOR-silencing line ( tor-es ) treated overnight with either DMSO (mock) or 5 μM β-estradiol. Western blotting was performed as in (B). (D) Co-immunoprecipitation of endogenous TOR with NPR1-GFP in transgenic Arabidopsis lines expressing 35S:NPR1-GFP or 35S:GFP . (E) Effects of SA on TOR activity and TOR-NPR1 interaction. 35S:NPR1-GFP seedlings were treated with mock or 0.3 mM SA for the indicated durations. The lysate was split into two portions: one was subjected to GFP immunoprecipitation followed by blotting with α-TOR and α-GFP; and the other was directly analyzed using α-TOR, α-pRPS6 (Ser-237), α-pRPS6 (Ser-240), α-RPS6, α-pS55/59, and α-GFP. Total protein loading was estimated using Ponceau S staining. *, nonspecific band. (F) Basal and SA-induced resistance in tor mutants. 3.5-week-old WT, tor-es #1 , and tor-es #2 were pretreated with mock or 10 μM β-estradiol 2 days before mock or 1 mM SA treatment. Psm (OD 600 = 0.001) was inoculated one day after mock or SA treatment. n = 8. (G) Effect of TOR inhibition on PR1 gene expression. RT-qPCR was performed on 35S:NPR1-GFP, 35S:npr1 S55/59A - GFP , and 35S:npr1 S55/59D - GFP seedlings pre-treated with mock or 2 μM AZD-8055 and subsequently treated with mock or 0.3 mM SA for 8 hours. (H) Effect of TOR inhibition on basal resistance. 3.5-week-old 35S:NPR1-GFP and 35S:npr1 S55/59D - GFP plants were pre-treated with mock or 2 μM AZD-8055 for 24 hours before inoculation with Psm (OD 600 = 0.0002). n = 8. (I) Proposed model of SnRK1- and TOR-mediated coordination between metabolic status and SA-induced immunity through differential phosphorylation of NPR1. Under optimal growth conditions (left), TOR is active (bright green) and promotes phosphorylation of NPR1 at Ser-55/59, thereby maintaining NPR1 in the cytoplasm through oligomerization, whereas SnRK1 remains inactive (dim red) under nutrient-rich conditions. Upon immune activation (right), SA, through an unknown mechanism, reduces glucose, Glc6P, and Tre6P levels, leading to activation of SnRK1 (bright red) and inhibition of TOR (dim green), accompanied by reduced Ser-55/59 phosphorylation. SA-induced redox changes also facilitate NPR1 translocation into the nucleus where SnRK1 phosphorylates NPR1 at Ser-557. This phosphorylation partially activates NPR1, promotes nSINC formation and interactions with transcriptional regulators, and primes subsequent phosphorylation at Ser-11/15, which is required for both full-scale defense gene induction and NPR1 turnover via sequential ubiquitination (Ub). Individual columns were compared using one-way ANOVA with Tukey’s post hoc test (F and H) or a two-tailed Student’s t-test (G), and the interactions were tested using two-way ANOVA (F-H). See also Figure S5 .

Journal: Cell

Article Title: Salicylic Acid Engages Central Metabolic Regulators SnRK1 and TOR to Govern Immunity by Differential Phosphorylation of NPR1

doi: 10.1016/j.cell.2026.09.011

Figure Lengend Snippet: (A and B) Effect of TOR inhibition on Ser-55/59 phosphorylation. (A) 35S:NPR1-GFP seedlings were treated with mock, 0.3 mM SA (6 hours), the TOR inhibitor Torin 2 (10 μM; 1 or 3 days), or AZD-8055 (2 μM; 1 or 3 days). (B) 35S:NPR1-GFP seedlings were treated with 2 μM AZD-8055 for the indicated durations. Phosphorylated and total NPR1 were detected using α-pS55/59 and α-GFP. (C) Effect of estradiol-induced silencing of TOR on Ser-55/59 phosphorylation. NPR1-GFP was transiently expressed in protoplasts derived from leaves of WT or the estradiol-inducible TOR-silencing line ( tor-es ) treated overnight with either DMSO (mock) or 5 μM β-estradiol. Western blotting was performed as in (B). (D) Co-immunoprecipitation of endogenous TOR with NPR1-GFP in transgenic Arabidopsis lines expressing 35S:NPR1-GFP or 35S:GFP . (E) Effects of SA on TOR activity and TOR-NPR1 interaction. 35S:NPR1-GFP seedlings were treated with mock or 0.3 mM SA for the indicated durations. The lysate was split into two portions: one was subjected to GFP immunoprecipitation followed by blotting with α-TOR and α-GFP; and the other was directly analyzed using α-TOR, α-pRPS6 (Ser-237), α-pRPS6 (Ser-240), α-RPS6, α-pS55/59, and α-GFP. Total protein loading was estimated using Ponceau S staining. *, nonspecific band. (F) Basal and SA-induced resistance in tor mutants. 3.5-week-old WT, tor-es #1 , and tor-es #2 were pretreated with mock or 10 μM β-estradiol 2 days before mock or 1 mM SA treatment. Psm (OD 600 = 0.001) was inoculated one day after mock or SA treatment. n = 8. (G) Effect of TOR inhibition on PR1 gene expression. RT-qPCR was performed on 35S:NPR1-GFP, 35S:npr1 S55/59A - GFP , and 35S:npr1 S55/59D - GFP seedlings pre-treated with mock or 2 μM AZD-8055 and subsequently treated with mock or 0.3 mM SA for 8 hours. (H) Effect of TOR inhibition on basal resistance. 3.5-week-old 35S:NPR1-GFP and 35S:npr1 S55/59D - GFP plants were pre-treated with mock or 2 μM AZD-8055 for 24 hours before inoculation with Psm (OD 600 = 0.0002). n = 8. (I) Proposed model of SnRK1- and TOR-mediated coordination between metabolic status and SA-induced immunity through differential phosphorylation of NPR1. Under optimal growth conditions (left), TOR is active (bright green) and promotes phosphorylation of NPR1 at Ser-55/59, thereby maintaining NPR1 in the cytoplasm through oligomerization, whereas SnRK1 remains inactive (dim red) under nutrient-rich conditions. Upon immune activation (right), SA, through an unknown mechanism, reduces glucose, Glc6P, and Tre6P levels, leading to activation of SnRK1 (bright red) and inhibition of TOR (dim green), accompanied by reduced Ser-55/59 phosphorylation. SA-induced redox changes also facilitate NPR1 translocation into the nucleus where SnRK1 phosphorylates NPR1 at Ser-557. This phosphorylation partially activates NPR1, promotes nSINC formation and interactions with transcriptional regulators, and primes subsequent phosphorylation at Ser-11/15, which is required for both full-scale defense gene induction and NPR1 turnover via sequential ubiquitination (Ub). Individual columns were compared using one-way ANOVA with Tukey’s post hoc test (F and H) or a two-tailed Student’s t-test (G), and the interactions were tested using two-way ANOVA (F-H). See also Figure S5 .

Article Snippet: NPR1-GFP was purified from the extracts using GFP-Trap agarose beads (Chromotek) following the manufacturer’s instructions.

Techniques: Phospho-proteomics, Activity Assay, Inhibition, Derivative Assay, Western Blot, Co-Immunoprecipitation Assay, Transgenic Assay, Expressing, Immunoprecipitation, Staining, Gene Expression, Quantitative RT-PCR, Activation Assay, Translocation Assay, Ubiquitin Proteomics, Two Tailed Test

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