primary antibodies against gfp  (Novus Biologicals)

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

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

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

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

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

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

Journal: Frontiers in Immunology

Article Title: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event

doi: 10.3389/fimmu.2026.1951506

Figure Lengend Snippet: Fluorescence microscopy of representative control (B6/J, Ccr2 RFP+/+ Cx3cr1 GFP+/+ , and R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 ) and SAU (R161H +/- x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 ) mouse retinas for RFP and GFP at 50 days of age. 40x magnification.

Article Snippet: Primary antibodies against GFP (cat no. NB600-597, mouse anti-GFP, Novus Biologicals, Centennial, CO, USA) and RFP (cat no. NBP3-25634, rabbit anti-RFP, Novus Biologicals) were diluted 1:200 with DPBS and applied to the retinas and held at 4°C for at least 12 hours in dark conditions then washed 3 x 5 minutes with DPBS.

Techniques: Fluorescence, Microscopy, Control

Journal: Frontiers in Immunology

Article Title: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event

doi: 10.3389/fimmu.2026.1951506

Figure Lengend Snippet: Flow cytometry distinguishes retinal MG from infiltrating macrophages. (A, B) Analysis of peripheral blood mononuclear cells for RFP and GFP expression showing the background levels of RFP and GFP fluorescence in B6/J mice (A) and the presence of RFP + , GFP + and RFP + GFP + cells in the circulation of Ccr2 RFP+/+ Cx3cr1 GFP+/+ mice (B) . The gating strategy for selecting CD45 + cells from B6/J mice is the same as shown for Ccr2 RFP+/+ Cx3cr1 GFP+/+ mice. (C) Representative analysis of a B6/J mouse showing background levels of RFP and GFP fluorescence in retinal CD45 + cells. Background fluorescence for GFP indicated as GFP neg cells. (D) Representative analysis of R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 retinas showing that a minimal number of all CD45 + cells (gate 1) express Ccr2 (RFP), and that a single population comprising most of the CD45 + cells and specifically the CD45 med cells (gate 2) express a high level of Cx3cr1 (GFP). Retinal CD45 + cells negative for GFP expression (GFP neg ) or with a low or intermediated level of GFP expression (GFP lo-med ) are indicated. (E) Representative analysis of R161H +/- x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 retinas with SAU showing a large increase in both CD45 med and CD45 hi cells with most of the RFP + cells found in the CD45 hi population as either RFP + GFP neg or RFP + GFP lo-med cells and that they are myeloid cells or lymphocytes. For all analyses; retinal MG indicated by red circles, flow cytometry done at 50 days of age, n = 4 for all groups of mice, and the mean percentage of cells in each quadrant for all mice analyzed is indicated.

Article Snippet: Primary antibodies against GFP (cat no. NB600-597, mouse anti-GFP, Novus Biologicals, Centennial, CO, USA) and RFP (cat no. NBP3-25634, rabbit anti-RFP, Novus Biologicals) were diluted 1:200 with DPBS and applied to the retinas and held at 4°C for at least 12 hours in dark conditions then washed 3 x 5 minutes with DPBS.

Techniques: Flow Cytometry, Analysis, Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event

doi: 10.3389/fimmu.2026.1951506

Figure Lengend Snippet: Analysis of R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 retinas for Ccr2 + (RFP + ) cells following ONC. (A) Representative fundoscopy of pre-ONC and day 10 post-ONC retinas. (B) Representative flow cytometry analysis of retinal CD45 med cells for RFP and GFP at day 10 post-ONC with mean percentage of CD45 med cells expressing GFP and RFP from all examined retinas indicated.

Article Snippet: Primary antibodies against GFP (cat no. NB600-597, mouse anti-GFP, Novus Biologicals, Centennial, CO, USA) and RFP (cat no. NBP3-25634, rabbit anti-RFP, Novus Biologicals) were diluted 1:200 with DPBS and applied to the retinas and held at 4°C for at least 12 hours in dark conditions then washed 3 x 5 minutes with DPBS.

Techniques: Analysis, Flow Cytometry, Expressing

Journal: Journal of Neuro-Oncology

Article Title: Patient-derived tissue cultures complement neurospheres for preclinical evaluation of AAV-mediated gene delivery in glioblastoma

doi: 10.1007/s11060-026-05807-w

Figure Lengend Snippet: Experimental workflow for generating patient-derived GBM tissue slice cultures (PDTC) and neurosphere cultures (PDNS). Fresh tumor tissue was sectioned into 350 μm slices using a tissue chopper. Tissue punches (2–3 mm) were cultured on membrane inserts at an air–liquid interface to establish PDTCs. Remaining tissue was mechanically and enzymatically dissociated and cultured under serum-free conditions to generate PDNS. AAV vectors encoding GFP were added to the culture medium, and transduction was monitored by live confocal microscopy. At endpoint, cultures were analyzed by immunohistochemistry for GFP expression, stem cell markers, and microenvironmental cell populations to assess vector tropism and biological effects

Article Snippet: For immunofluorescence staining, samples were incubated overnight with primary antibodies ( stem cell marker : SOX2, Neuromics, GT15098 (1:200); Nestin, Merck, ABD69 (1:1000); OLIG2, Epitomics, AC-0106RUO (1:200); CD44, Cell Signaling Technologies, 156-3C11 (1:400); Survivin, Cell Signaling Technologies, 2808 (1:500) astrocyte marker : GFAP, DAKO, Z0334 (1:500); AAV transduction : GFP, Synaptic Systems, 132005 (1:400); apoptosis and proliferation : Cleaved PARP1, abcam, ab32064 (1:100), Ki67, DCS, KI681C01 (1:200)).

Techniques: Derivative Assay, Tissue, Cell Culture, Membrane, Transduction, Confocal, Microscopy, Immunohistochemistry, Expressing, Stem Cell, Plasmid Preparation

Journal: Journal of Neuro-Oncology

Article Title: Patient-derived tissue cultures complement neurospheres for preclinical evaluation of AAV-mediated gene delivery in glioblastoma

doi: 10.1007/s11060-026-05807-w

Figure Lengend Snippet: AAV2 and AAV6 transduction of patient-derived neurosphere cultures (PDNS) under EGF-supplemented and EGF-deprived conditions. ( A ) Dose-response analysis of AAV-mediated transduction in PDNS. Spheroids derived from at least three independent GBM specimens were transduced with AAV2 or AAV6 vectors at doses ranging from 10 6 to 10 9 vector particles per well. GFP expression was monitored by live confocal microscopy on days 2 and 5 post-transduction. Quantification of GFP fluorescence demonstrated a dose-dependent increase in transduction efficiency for both serotypes. For AAV6, significant increases in GFP signal were observed at 10 8 particles under EGF-supplemented conditions, whereas AAV2 reached significance only at 10 9 particles on day 5. Representative confocal images of AAV6-transduced spheroids cultured in the presence of EGF are shown. To validate live-imaging results and determine cellular tropism, spheroids were fixed on day 5, embedded, and analyzed by immunofluorescence staining for GFP. ( B ) PLAT-normalized AAV vector genome abundance in PDNS 5 days after treatment with 10⁸ vector particles is shown ( n = 4). ( C ) Cell type–specific analysis of AAV transduction in PDNS. Co-immunofluorescence staining for GFP and the lineage-associated markers GFAP, CD44, Nestin, and SOX2 was performed to identify transduced cellular populations. In EGF-deprived media, increased proportions of GFP-positive cells were observed across multiple marker-defined populations. In contrast, under EGF-supplemented conditions, the strongest enrichment of GFP expression was detected within the Nestin-positive population. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s multiple-comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001)

Article Snippet: For immunofluorescence staining, samples were incubated overnight with primary antibodies ( stem cell marker : SOX2, Neuromics, GT15098 (1:200); Nestin, Merck, ABD69 (1:1000); OLIG2, Epitomics, AC-0106RUO (1:200); CD44, Cell Signaling Technologies, 156-3C11 (1:400); Survivin, Cell Signaling Technologies, 2808 (1:500) astrocyte marker : GFAP, DAKO, Z0334 (1:500); AAV transduction : GFP, Synaptic Systems, 132005 (1:400); apoptosis and proliferation : Cleaved PARP1, abcam, ab32064 (1:100), Ki67, DCS, KI681C01 (1:200)).

Techniques: Transduction, Derivative Assay, Analysis, Spheroids, Plasmid Preparation, Expressing, Confocal, Microscopy, Fluorescence, Cell Culture, Imaging, Immunofluorescence, Staining, Marker, Comparison

Journal: Journal of Neuro-Oncology

Article Title: Patient-derived tissue cultures complement neurospheres for preclinical evaluation of AAV-mediated gene delivery in glioblastoma

doi: 10.1007/s11060-026-05807-w

Figure Lengend Snippet: Comparison of AAV-mediated transduction and stem cell marker expression in patient-derived tissue cultures (PDTCs) and neurosphere cultures (PDNS). ( A ) Quantitative comparison of AAV transduction in matched PDTC and PDNS generated from seven independent GBM specimens (shown is the median). GFP fluorescence intensity was used as a readout of AAV-mediated gene delivery following treatment with AAV2 or AAV6 under EGF-supplemented and EGF-deprived conditions. PDTCs exhibited greater inter-patient variability than PDNS across all experimental conditions. In PDTCs, both AAV2 and AAV6 significantly increased GFP expression compared with control cultures. In contrast, only AAV6 induced a significant increase in GFP fluorescence in PDNS, whereas AAV2 did not differ significantly from control conditions. EGF supplementation had minimal effects on transduction efficiency in PDTCs, while omission of EGF reduced GFP expression in PDNS, particularly following AAV6 transduction. ( B ) GFP expression could also be observed in PDTC of non-malignant origin in both serotypes investigated in two independent tissue specimen. ( C ) Expression of stem cell-associated markers following AAV transduction in PDTCs and PDNS. Nestin expression remained stable across culture systems and treatment conditions. In contrast, SOX2 expression exhibited culture- and treatment-dependent variability. A significant increase in SOX2 expression was observed in PDNS transduced with AAV6 under EGF-deprived conditions compared with untreated controls, whereas no significant alterations were detected in PDTCs. These findings indicate that SOX2 expression varied with culture and treatment conditions in PDNS, whereas no significant changes were detected in PDTCs. Statistical analyses were performed using the Kruskal–Wallis test followed by Dunn’s multiple-comparison test (* p < 0.05, ** p < 0.01, **** p < 0.0001). Individual data points represent independent patient specimens

Article Snippet: For immunofluorescence staining, samples were incubated overnight with primary antibodies ( stem cell marker : SOX2, Neuromics, GT15098 (1:200); Nestin, Merck, ABD69 (1:1000); OLIG2, Epitomics, AC-0106RUO (1:200); CD44, Cell Signaling Technologies, 156-3C11 (1:400); Survivin, Cell Signaling Technologies, 2808 (1:500) astrocyte marker : GFAP, DAKO, Z0334 (1:500); AAV transduction : GFP, Synaptic Systems, 132005 (1:400); apoptosis and proliferation : Cleaved PARP1, abcam, ab32064 (1:100), Ki67, DCS, KI681C01 (1:200)).

Techniques: Comparison, Transduction, Stem Cell, Marker, Expressing, Derivative Assay, Tissue, Generated, Fluorescence, Control