rabbit polyclonal atf4 antibody  (Bioss)

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