polyclonal detection antibodies  (nanoComposix)

Journal: Chemical Science

Article Title: A (poly)Pro tip for preserving native disulfide connectivity during thiol–maleimide bioconjugation of disulfide-rich peptides

doi: 10.1039/d5sc08821f

Figure Lengend Snippet: Crystal structure of tetrameric ectodomain of LINGO-1 (left). Structure of the LINGO-1 N-cap [1–20] region, displaying a DRP motif (top right, cornflower blue). 3D NMR solution structure obtained for synthetic LINGO-1[1–20], 1 (box, bottom right, magenta).

Article Snippet: Remarkably, our polyclonal IgG detected LINGO-1 at a five-fold higher dilution than the commercial polyclonal IgG antibody AF3086 In a previous study, screening of all commercially available IgGs identified polyclonal IgG AF3086 as the most suitable for co-immunoprecipitation studies. (R&D Systems), while also yielding stronger overall staining intensity (Fig. S52 and S53).

Techniques:

Journal: Chemical Science

Article Title: A (poly)Pro tip for preserving native disulfide connectivity during thiol–maleimide bioconjugation of disulfide-rich peptides

doi: 10.1039/d5sc08821f

Figure Lengend Snippet: Synthesis of C-terminal amidated LINGO-1[1–20]-Gly-Anl 4. Reagents and conditions: [a] DMSO/H 2 O – 1 : 9, peptide conc. 0.32 mM*, 100 h, r.t.; [b] AcOH/160 mM aq. HCl – 93.6 : 6.4 containing 62.5 mM of I 2 , peptide conc. 1 mM*, 2 h, r.t. Abbreviations: Anl = 6-Azido- l -norleucine, represented in the scheme as “K(N 3 )”; DMSO = dimethyl sulfoxide; r.t. = room temperature. *Steps [a] and [b] were carried out on crude material, with concentrations calculated from the crude mass as if it corresponded to the pure compound.

Article Snippet: Remarkably, our polyclonal IgG detected LINGO-1 at a five-fold higher dilution than the commercial polyclonal IgG antibody AF3086 In a previous study, screening of all commercially available IgGs identified polyclonal IgG AF3086 as the most suitable for co-immunoprecipitation studies. (R&D Systems), while also yielding stronger overall staining intensity (Fig. S52 and S53).

Techniques:

Journal: Chemical Science

Article Title: A (poly)Pro tip for preserving native disulfide connectivity during thiol–maleimide bioconjugation of disulfide-rich peptides

doi: 10.1039/d5sc08821f

Figure Lengend Snippet: (A) ELISA cell assay showing the interaction between decreasing concentrations of polyclonal IgG and HEK cells expressing LINGO-1. Absorbance values at 450 nm were background-corrected by subtracting the signal from non-transfected HEK cells. (B) Superposition of the [1–20] region for LINGO-1-to-4 homologs shows that all Cys residues are conserved; additional conserved residues are highlighted in dark grey, whereas similar residues—defined by comparable hydrophilicity ( e.g. , Ser vs. Thr) or charge ( e.g. , Lys, Arg, or His)—are shown in light grey. (C) Western blot detail in which polyclonal IgG was used for staining HEK cells and LINGO homologs 1–4. Anti-GAPDH staining is shown at the bottom as a loading control.

Article Snippet: Remarkably, our polyclonal IgG detected LINGO-1 at a five-fold higher dilution than the commercial polyclonal IgG antibody AF3086 In a previous study, screening of all commercially available IgGs identified polyclonal IgG AF3086 as the most suitable for co-immunoprecipitation studies. (R&D Systems), while also yielding stronger overall staining intensity (Fig. S52 and S53).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Western Blot, Staining, Control

Journal: Nature Communications

Article Title: Investigational eIF2B activator DNL343 modulates the integrated stress response in preclinical models of TDP-43 pathology and individuals with ALS in a randomized clinical trial

doi: 10.1038/s41467-025-63031-y

Figure Lengend Snippet: a Schematic depicting the ISR pathway, including kinases mediating the phosphorylation of eIF2α, and activation of eIF2B by DNL343. b Confocal microscopy images showing induced expression of GFP-TDP-43 FL and GFP-TDP-43 (86-414) variant in H4 cells increased immunoreactivity of ATF4 (anti-ATF4, red) compared to GFP control. This ATF4 signal was reduced with 1 µM DNL343 treatment. Scale bar: 20 µm. c Quantification of ATF4 immunoreactivity in ( b ) from n = 3 (GFP Control) or 4 biological replicates (GFP-TDP-43 variants). a.u.: arbitrary units. d ECLIA-based assay result demonstrating elevated ATF4 protein levels with induced expression of GFP-TDP-43 FL and GFP-TDP-43 (86-414) , compared to GFP control cells. Pre-treatment with DNL343 reduced ATF4 protein levels in each cell line ( n = 6 biological replicates). e Volcano plot of differentially regulated genes in H4 cells induced with GFP-TDP-43 FL compared to GFP control. Representative ISR genes with significant expression changes are labeled as red (upregulation) or blue (downregulation) and insignificant genes as gray. Significance cutoff, adjusted p -value < 0.05. f Volcano plot of differentially regulated genes in H4 cells induced with GFP-TDP-43 (86-414) compared to GFP control. Representative ISR genes are labeled as in ( e ). Significance cutoff, adjusted p -value < 0.05. g Gene set enrichment analysis (GSEA) plot for ISR gene comparing truncated GFP-TDP-43 (86-414) from GFP control expressing cells. h Volcano plot of differentially regulated genes 24 hours after DNL343 treatment in H4 cells induced with GFP-TDP-43 (86-414) . The same set of representative ISR genes from ( e ) and ( f ) are labeled. All data are shown as mean ± SEM ( c , d ). Statistical significance was determined with one-way ANOVA, with Tukey’s multiple comparison, ns P > 0.05. Source data are provided in Source Data file.

Article Snippet: Plates were washed as above and 25 μL of 0.5 μg/mL Rabbit anti-ATF4 Sulfo Tag polyclonal detection antibody in 1X MSD Blocker A (MSD, #R93AA-2) + 0.1 mg/mL Rabbit Gamma Globulin (Rockland Immunochemicals, #D610-1000) + 1 mg/mL Mouse Gamma Globulin (Rockland Immunochemicals, #D609-0100) in 1x TBST was added to each well.

Techniques: Phospho-proteomics, Activation Assay, Confocal Microscopy, Expressing, Variant Assay, Control, Labeling, Comparison

Journal: Nature Communications

Article Title: Investigational eIF2B activator DNL343 modulates the integrated stress response in preclinical models of TDP-43 pathology and individuals with ALS in a randomized clinical trial

doi: 10.1038/s41467-025-63031-y

Figure Lengend Snippet: a Experimental design of acute DN9058 dosing of rNLS8 transgenic mice. All animals were fed Dox-containing diet until 8 weeks of age, including non-transgenic (nTg) controls, single transgenic (sTg) controls and double transgenic (rNLS8) mice, then Dox was removed from their diet except for group 6 (double transgenic (rNLS8) on Dox for two additional weeks). On 13th and 14th day off Dox, indicated groups were dosed with DN9058 by oral gavage at 50 mg/kg per animal weight. b - c Quantification of p-eIF2α normalized to loading control eIF2α ( b ) and ATF4 level normalized to GAPDH ( c ) from Supplementary Fig. and j ( n = 8 sTg Control or 12 rNLS8 mice). Data are shown as fold-changes relative to vehicle-treated control mice. d Transcriptional fold change of pre-selected ISR genes in rostral cortex of indicated mouse line ( n = 7 (nTg Ctrl), 8 (rNLS8 Dox), 9 (sTg Ctrl) and 12 (rNLS8) mice). Data are shown as mean ± SEM ( b − d ). Statistical significance was determined with Kruskal-Wallis test with Dunn’s multiple comparisons ( b ), ordinary One-way ANOVA with Tukey’s multiple comparison ( c , d ). Source data are provided in Source Data file.

Article Snippet: Plates were washed as above and 25 μL of 0.5 μg/mL Rabbit anti-ATF4 Sulfo Tag polyclonal detection antibody in 1X MSD Blocker A (MSD, #R93AA-2) + 0.1 mg/mL Rabbit Gamma Globulin (Rockland Immunochemicals, #D610-1000) + 1 mg/mL Mouse Gamma Globulin (Rockland Immunochemicals, #D609-0100) in 1x TBST was added to each well.

Techniques: Transgenic Assay, Control, Comparison

Journal: Nature Communications

Article Title: Investigational eIF2B activator DNL343 modulates the integrated stress response in preclinical models of TDP-43 pathology and individuals with ALS in a randomized clinical trial

doi: 10.1038/s41467-025-63031-y

Figure Lengend Snippet: a Experimental design of chronic DN9058 dosing for 6 weeks off dox (WOD). DN9058 was formulated at 50 mg/kg per chow for the chronic administration to the indicated conditions. b - c Quantification of p-eIF2α normalized to loading control eIF2α ( b ) and ATF4 level normalized to GAPDH ( c ) from Supplementary Fig. and j, respectively ( n = 8 (sTg Control), 10 (rNLS8 Veh), and 13 (rNLS8 DN9058) mice). Data are shown as fold-changes relative to vehicle-treated control mice. d - e Transcript level changes of pre-selected ISR genes ( d ) and ISR genes indicated in later stage of the disease ( e ) in rostral cortex ( n = 9 (sTg Control), 10 (rNLS8 Dox), 11 (rNLS8 Veh), and 16 (rNLS8 DN9058) mice). f Time (weeks) to show clasping for individual rNLS8 mice that demonstrated the clasping phenotype ( n = 13 (Veh) and 18 (DN9058) mice). g Plasma NfL concentration at baseline (0 WOD), 2, 4, and 6 WOD in rNLS8 mice ( n = 11 (Veh) and 16 (DN9058) mice). The same figure legend for rNLS8 + Veh (yellow) and rNLS8 + DN9058 (orange) is shared with panel ( f ). Source data are provided in Source Data file. Data are shown as mean ± SEM ( b − e ) and individual values overlayed on violin plot ( f , g ). Statistical significance was determined with Kruskal-Wallis test with Dunn’s multiple comparisons ( b , c ), ordinary One-way ANOVA with Tukey’s multiple comparison ( d , e ), Two-tailed Mann-Whitney test ( f ), and Two-way ANOVA with multiple comparisons ( g ).

Article Snippet: Plates were washed as above and 25 μL of 0.5 μg/mL Rabbit anti-ATF4 Sulfo Tag polyclonal detection antibody in 1X MSD Blocker A (MSD, #R93AA-2) + 0.1 mg/mL Rabbit Gamma Globulin (Rockland Immunochemicals, #D610-1000) + 1 mg/mL Mouse Gamma Globulin (Rockland Immunochemicals, #D609-0100) in 1x TBST was added to each well.

Techniques: Control, Clinical Proteomics, Concentration Assay, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Investigational eIF2B activator DNL343 modulates the integrated stress response in preclinical models of TDP-43 pathology and individuals with ALS in a randomized clinical trial

doi: 10.1038/s41467-025-63031-y

Figure Lengend Snippet: a − d ATF4 protein ( a ) and CHAC1 gene ( b ) expression in ex vivo stimulated PBMCs from healthy participants. ATF4 protein ( c ) and CHAC1 gene ( d ) expression in ex vivo stimulated PBMCs from ALS participants. PBMCs were freshly isolated from participants in each dose group shown at the times indicated and analyzed by either ECLIA ( a , c ) or multiplex qPCR ( b , d ). Values shown as median (IQR = interval from the first to the third quartile, shown as error bars) percent change from baseline. For MAD healthy participants ATF4 protein, n = 12, 6, 7, 7, 7, 6 participants for placebo, 45 mg, 100 mg, 145 mg, 200 mg and 260 mg groups respectively; CHAC1 gene, n = 11, 6, 7, 5, 7, 6 participants for placebo, 45 mg, 100 mg, 145 mg, 200 mg and 260 mg groups respectively. For ALS participants ATF4 protein, n = 6, 7, 6 participants for placebo, 100 mg, and 200 mg groups respectively; CHAC1 gene, n = 5, 7, 6 participants for placebo, 100 mg, 200 mg groups respectively through Day 28 for each dose group. e Heat map depicting relative change from baseline for a panel of ISR genes. Gene expression measured by multiplex qPCR in freshly isolated ex vivo stimulated PBMCs from ALS patients in each dose group. Values grouped based on median percent change from baseline and genes rank ordered based on percent change from baseline at Day 28 in the highest dose cohort (200 mg). f Percent change from baseline in CSF GDF-15 protein concentration at Day 28 in ALS participants, n = 9, 8, 8 participants for placebo, 100 mg, 200 mg groups respectively per group. Data are shown as box plot with individual percent change values overlayed. The middle line of the boxplot displays the median, the lower and upper limits of the box display the first and third quartiles, and the whiskers extend to the largest and smallest values. Source data are provided in Source Data file.

Article Snippet: Plates were washed as above and 25 μL of 0.5 μg/mL Rabbit anti-ATF4 Sulfo Tag polyclonal detection antibody in 1X MSD Blocker A (MSD, #R93AA-2) + 0.1 mg/mL Rabbit Gamma Globulin (Rockland Immunochemicals, #D610-1000) + 1 mg/mL Mouse Gamma Globulin (Rockland Immunochemicals, #D609-0100) in 1x TBST was added to each well.

Techniques: Expressing, Ex Vivo, Isolation, Multiplex Assay, Gene Expression, Protein Concentration