rabbit polyclonal anti atf4  (Proteintech)

Journal: Virologica Sinica

Article Title: Modulation of the unfolded protein response by white spot syndrome virus via wsv406 targeting BiP to facilitate viral replication

doi: 10.1016/j.virs.2024.10.005

Figure Lengend Snippet: Knockdown of wsv406 downregulates PERK-eIF2α pathway during WSSV infection in vivo . A The phosphorylation of eIF2α was detected in hemocyte (left panel) and gill (right panel) at 48 ​h post WSSV infection by western blotting. Beta-actin was used as a loading control to calculate the ratio of p -eIF2α/eIF2α. VP28 served as the indicator for infection. B LvATF4 nuclear translocation was inhibited in wsv406-silenced hemocytes. The Image J software calculated the percentage of ATF4 into the nucleus. C Viral genes expression levels in hemocyte (left panel) and gill (right panel) of dswsv406-treated shrimp at 48 ​h post WSSV infection were detected by qRT-PCR. The data were provided as the means ​± ​SD of triplicate assays and analyzed statistically by Student's t -test ( ∗∗ P ​< ​0.01).

Article Snippet: The following steps were the same as above, the primary antibodies used were rabbit anti-ATF4 antibody (Bioss, bs-1531R), rabbit anti-ATF6 antibody (Bioss, bs-1634R) and mouse anti-β-actin antibody (Sigma, A1978).

Techniques: Knockdown, Infection, In Vivo, Western Blot, Control, Translocation Assay, Software, Expressing, Quantitative RT-PCR

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

Journal: JCI insight

Article Title: The PERK/ATF4 pathway is required for metabolic reprogramming and progressive lung fibrosis.

doi: 10.1172/jci.insight.189330

Figure Lengend Snippet: Figure 5. PERK activates PGC-1α by increasing ATF4 in lung macrophages. (A) Schematic representation of PERK pathway activation and downstream signaling molecules. CHOP, C/EBP homologous protein. (B) Lung macrophages from normal and asbestosis humans were obtained by BAL. Densitom- etry of immunoblot. Inset, immunoblot analysis of PGC-1α (n = 3). (C) Macrophages were cotransfected with renilla luciferase plasmid, pGL3-Ppargc1a luciferase promoter, and empty, PERKWT, or PERKDN and exposed to asbestos (24 hours). Ppargc1a promoter activity was determined by measuring firefly and renilla luciferase (n = 3). (D) ATF3 (n = 3), (E) ATF4 (n = 3), and (F) PPARGC1A mRNA expression (n = 3) in transfected macrophages exposed to asbestos. (G) Macrophages were exposed to control or asbestos and subjected to ChIP with antibodies against ATF3 or ATF4 followed by real-time PCR to determine Ppargc1a promoter binding (n = 3–9). (H) Lung macrophages were obtained from normal and asbestosis humans by BAL. Densitome- try of immunoblot. Inset, immunoblot analysis of ATF4 (n = 4). (I) Macrophages were cotransfected with pGL3-Ppargc1a luciferase promoter combined with scramble or ATF4 siRNA, and empty or PERKWT, and exposed to asbestos. Ppargc1a promoter activity (n = 3–4). Inset, immunoblot analysis for ATF4. (J) Schematic illustration of ATF4 binding site on Ppargc1a promoter in the cAMP response element (CRE) domain and mutation sites on specific residues. (K) Ppargc1a promoter luciferase activity in macrophages transfected with empty, PERKWT, or pGL3-Ppargc1a luciferase mutant and exposed to asbestos (n = 3). (L) Ppargc1a mRNA expression in BAL isolated at day 21 from exposed Eif2ak3fl/fl and Eif2ak3–/– Lyz2-cre mice (n = 4). (M) Ppargc1a

Article Snippet: Briefly cross-linked chromatin preparations were used for input controls (2% of total) or for immunoprecipitation of ATF3 (D2Y5W) anti-rabbit monoclonal (33593; Cell Signaling Technology), ATF4 anti-rabbit polyclonal (10835-1-AP; Proteintech), histone H3 (D1H2) XP Rabbit (4499; Cell Signaling Technology) as a positive control, or normal rabbit IgG (2729; Cell Signaling Technology) as a negative control.

Techniques: Activation Assay, Western Blot, Luciferase, Plasmid Preparation, Activity Assay, Expressing, Transfection, Control, Real-time Polymerase Chain Reaction, Binding Assay, Mutagenesis, Isolation

Journal: JCI insight

Article Title: The PERK/ATF4 pathway is required for metabolic reprogramming and progressive lung fibrosis.

doi: 10.1172/jci.insight.189330

Figure Lengend Snippet: Figure 7. Pharmacological inhibition of PERK abrogates FAO in mice with established fibrosis. Thirteen days after exposure, GSK (30 mg/kg i.p.) was administered daily to mice. (A) FAO in BAL macrophages from asbestos- or MMVF-exposed mice was measured by OCR with the addition of BSA or BSA:palmitate (PMT) using Seahorse XF96 bioanalyzer (Agilent Technologies) (n = 4–5). min-μg, minute/μg protein. Total RNA was isolated from lung macrophages and subjected to real-time PCR for (B) Ppargc1a (n = 3) and (C) Atf4 mRNA expression (n = 3). (D) FAO in macrophages from control or bleo- mycin-exposed mice was measured by OCR with the addition of BSA or PMT using Seahorse XF96 bioanalyzer (n = 3–5). Total RNA was isolated from lung macrophages and subjected to real-time PCR for (E) Ppargc1a (n = 3) and (F) Atf4 mRNA expression (n = 3). Data shown as mean ± SEM. One-way ANOVA with Tukey’s post hoc comparison in B and C. Two-tailed Student’s t test in E and F. **P ≤ 0.01, ****P ≤ 0.0001. (See also Supplemental Figure 6.)

Article Snippet: Briefly cross-linked chromatin preparations were used for input controls (2% of total) or for immunoprecipitation of ATF3 (D2Y5W) anti-rabbit monoclonal (33593; Cell Signaling Technology), ATF4 anti-rabbit polyclonal (10835-1-AP; Proteintech), histone H3 (D1H2) XP Rabbit (4499; Cell Signaling Technology) as a positive control, or normal rabbit IgG (2729; Cell Signaling Technology) as a negative control.

Techniques: Inhibition, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control, Comparison, Two Tailed Test

Journal: Cell reports

Article Title: Thbs1 regulates skeletal muscle mass in a TGFβ-Smad2/3-ATF4-dependent manner

doi: 10.1016/j.celrep.2024.114149

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-ATF4 , LSBio (LifeSpan) , Cat# LS-B6361; RRID:AB_11042790.

Techniques: Virus, Recombinant, Electron Microscopy, Plasmid Preparation, SYBR Green Assay, Membrane, Blocking Assay, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Protein Extraction, Cloning, Gene Expression, Software, Microscopy, Real-time Polymerase Chain Reaction