anti foxo3  (Cell Signaling Technology Inc)

Journal: Poultry Science

Article Title: Comparative transcriptomic analysis reveals breed-specific developmental characters of skin and feather follicles between Anser Cygnoides and Anser Anser

doi: 10.1016/j.psj.2025.105457

Figure Lengend Snippet: Immunohistochemistry staining of FOXO3 distrubutions in in embryonic skin and feather follicles of Zhedong and Hungarian white geese as detected by IHC. Bar = 50 or 200 μm .

Article Snippet: The sections were then incubated overnight at 4°C with 50 μL of rabbit anti-FOXO3 antibody (Cat# WL02891, WANLEIBIO, Shenyang, China) diluted at 1:500, rabbit anti-SOX10 antibody (Cat# WL05169, WANLEIBIO, Shenyang, China) diluted at 1:500, and rabbit anti-β-catenin antibody (Cat# WL0962a, WANLEIBIO, Shenyang, China) at 1:500 diluent.

Techniques: Immunohistochemistry, Staining

Journal: eLife

Article Title: Aurora kinase A promotes trained immunity via regulation of endogenous S-adenosylmethionine metabolism

doi: 10.7554/eLife.104138

Figure Lengend Snippet: ( A ) Protein level of GNMT in bone marrow-derived macrophages (BMDMs) with FOXO3 knockdown was detected by Western blot; * showed the position of FOXO3 band. ( B, C ) Western blot analysis of GNMT downregulation by si Foxo3 in trained BMDMs with AurA inhibition. BMDMs were transfected with siRNA targeting FOXO3 for 48 hr, followed by β-glucan (50 μg/mL) and alisertib (1 μM) for 24 hr ( B ); BMDMs were co-transfected with siRNAs targeting FOXO3 and AurA for 48 hr, followed by β-glucan (50 μg/mL) stimulation for another 24 hr ( C ). ( D ) Supernatant levels of IL-6 in BMDMs. The cells were treated with β-glucan (50 μg/mL) and alisertib (1 μM) after transfection of siRNAs targeting FOXO3. ( E ) Western blot analysis of phosphorylation level of FOXO3 at ser 315 in BMDMs treated with β-glucan (50 μg/mL) with or without alisertib (1 μM) for 12 hr. ( F ) Immunofluorescence staining of FOXO3 in BMDMs after 12 hr β-glucan stimulation with or without alisertib. Scale bars: 10 μm (left). The nuclear localization of FOXO3 was compared by calculating the ratio of mean nuclear intensity to cytoplasmic intensity, and the representative data (right) showed the mean intensity of counted macrophages. ( G ) Western blot analysis of AKT-mTOR-S6 pathway in β-glucan-trained BMDM. BMDMs were transfected with siRNA targeting AurA for 48 hr, followed by β-glucan stimulation for 6 hr (left); BMDM was trained with β-glucan in the absence or presence of alisertib for 6 hr (right). ( H ) Supernatant levels of IL-6 and TNF in BMDMs. The trained cells were treated with siRNA targeting AurA or alisertib in combination with an mTOR agonist, MHY1485 (2 μM), and restimulated with MC38 culture supernatant for 48 hr. Data in D and H are representative of three independent experiments and presented as the mean ± SEM. P values were derived from one-way ANOVA with Tukey’s multiple-comparison test compared with each other in D and H , or with Dunnett’s multiple-comparison test in F compared with β-glucan only group. In A-C and E-G , similar results were obtained for three independent experiments. Related to . Figure 5—source data 1. Uncropped and labeled blots for . Figure 5—source data 2. Raw unedited blots for . Figure 5—source data 3. Original images for .

Article Snippet: Antibody , Rabbit polyclonal anti-Phospho FOXO3 (Ser315) , Proteintech , Cat# 28755–1-AP, RRID: AB_2881210 , IB (1:500).

Techniques: Derivative Assay, Knockdown, Western Blot, Inhibition, Transfection, Phospho-proteomics, Immunofluorescence, Staining, Comparison, Labeling

Journal: Experimental Hematology & Oncology

Article Title: A one-base therapeutic insertion in the HBG2 distal promoter reactivates γ-globin expression

doi: 10.1186/s40164-025-00626-7

Figure Lengend Snippet: InsA mutation creates a de novel site for activator FOXO3. ( a ) The binding motifs of FOXO3 were identified using JASPAR. ( b ) Dual luciferase was performed to determine the interaction between FOXO3 and insA. The wild type (WT) or insA- HBG promoter were cloned into the pGL4.18 vector and co-transfected with FOXO3 of pcDNA3.1 into HUDEP-2 cells. ( c ) EMSA was performed with insA or wild type (WT) hot probe, and indicated fold molar excess of the insA or WT cold probe in HUDEP-2 nuclear extract. Anti-FOXO3 antibody was used to perform super shift-EMSA. ( d ) ChIP-seq analysis with anti-FOXO3 antibody in WT, insA and insA KO HUDEP-2 clones. The light green shadow indicated the binding peak of FOXO3 on the HBG2 distal promoter. ( e ) ChIP-qPCR analysis with anti-FOXO3 antibody in control (Ctrl) and insA HUDEP-2 cells. Data from > 3 independent experiments are presented as mean ± SD (** p < 0.01). ( f - h ) Western blot analysis in FOXO3 knock out (KO) HUDEP-2 control ( f ) or insA clones ( g ) on day 8 of the differentiation, and in FOXO3 overexpression (OE) in insA HUDEP-2 clone ( h ) without differentiation. ( i - j ) The expression level of IRS1 , PIK3R1 , PDK2 and AKT1 measured by RNA-seq ( i ) and qPCR ( j ) in control, insA and insA KO clones. Data from > 3 independent experiments are presented as mean ± SD (* P < 0.05, ** p < 0.01, *** P < 0.001, ns., no significant). ( k ) Western blot analysis with anti-phosphorylated AMPK (p-AMPK), anti-phosphorylated AKT and FOXO3 ser413 antibodies in control (Ctrl) and insA pool or clone HUDEP-2 cells. GAPDH served as control. ( l ) Gene ontology (GO) analysis of the identified proteins pulling down by anti-FOXO3 ser413 antibody in control and insA HUDEP-2 cells. BP, biological process; CC, cellular component; MF, molecular function

Article Snippet: The primary antibodies were as follow: anti-HbF (ab137096, Abcam), anti-GAPHD (129-10312, Ray antibody), anti-lamin A/C (ab238303, Abcam), anti-β-Tublin (ab6046, Abcam); anti-phospho-FOXO3 (Ser413) (8174s), anti-phospho-AMPK (Thr172) (2535T), all from Cell Signaling Technology.

Techniques: Mutagenesis, Binding Assay, Luciferase, Clone Assay, Plasmid Preparation, Transfection, ChIP-sequencing, ChIP-qPCR, Control, Western Blot, Knock-Out, Over Expression, Expressing, RNA Sequencing