mouse mab against foxo1  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a-b Immunoblot (IB) analysis of lung tissues (LT) showed increased expression of Akt1 and decreased expression of FoxO1 in Chfr ΔEC as compared to Chfr fl/fl (WT) mice ( n =4 mice/genotype). Shown are mean values ± SEM (unpaired two-tailed Student’s t test). C Chfr fl/fl and Chfr ΔEC mice injected with LPS (10 mg/kg bodyweight, i.p.) for 0, 6, and 24 h were used to measure the expression of Akt1 in lung tissue. Shown are mean values ± SEM ( n = 3 mice/genotype/group; two-way ANOVA followed by Tukey’s post-hoc test). d-e Chfr fl/fl and Chfr ΔEC mice were i.p. injection of LPS (10 mg/kg bodyweight) for 0, 6, and 24 h. Thereafter, lung tissues harvested were used for RT-qPCR to measure the mRNA expression of Ang-2 ( d ) or IB analysis was performed to determine the expression of Ang-2 and Tie-2 ( e ). Shown are mean values ± SEM ( n = 3 mice/genotype/group; two-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Mouse mAb against Akt1 (catalog #sc-5298; IB, 1:1000; IP, 1 μg/100 μg cell lysate protein), and mouse mAb against FoxO1 (catalog #sc-374427; IS, 1:100) were from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Two Tailed Test, Injection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HLMVEC were transfected with Sc-siRNA or CHFR-siRNA. At 72 h post-transfection, cells challenged with LPS (5 μg/ml) for different time periods were used for IB analysis to determine expression of CHFR and Akt1. Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test). b-c HLMVEC were transfected with Sc-siRNA or CHFR-siRNA. At 72 h post transfection, cells challenged with LPS (5 μg/ml) for different time periods were stained with antibodies specific to VE-cadherin, Akt1, and FoXO1. Confocal imaging shows that CHFR depletion in EC prevents LPS-induced degradation of Akt1 and VE-cadherin and nuclear accumulation of FoxO1. d CHFR depletion prevents LPS-induced expression of FoxO1 and Ang-2. HLMVEC were transfected with Sc-siRNA or CHFR-siRNA as above and exposed to LPS for different time periods were used for IB to determine expression of FoxO1 and Ang-2. e LPS-induced time-course expression of FoxO1, CHFR, and Ang-2 in HLMVEC. HLMVEC exposed to LPS (5 μg/ml) for 0, 1, 2, 4, and 6 h were used for IB. d-e Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Mouse mAb against Akt1 (catalog #sc-5298; IB, 1:1000; IP, 1 μg/100 μg cell lysate protein), and mouse mAb against FoxO1 (catalog #sc-374427; IS, 1:100) were from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Staining, Imaging

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a-d HLMVEC were pretreated with the Akt1 inhibitor (Akt1-inh) for 1 h before stimulation with LPS (5 μg/ml) for 0, 6, 12, and 24 h. IB analysis showed increased expression of FoxO1, Ang-2, and CHFR, whereas expression of Akt1 and VE-cadherin was suppressed. Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post hoc test).

Article Snippet: Mouse mAb against Akt1 (catalog #sc-5298; IB, 1:1000; IP, 1 μg/100 μg cell lysate protein), and mouse mAb against FoxO1 (catalog #sc-374427; IS, 1:100) were from Santa Cruz Biotechnology.

Techniques: Expressing

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HMEC were transfected with WT or K/R-mutant Akt1 constructs. At 36 h after transfection, cells were treated with LPS (5 μg/ml) for 0, 12, and 24h and then cells were used for IB to determine expression of Akt1 and VE-cadherin. Shown are mean values ± SEM (n = 3 independent experiments; two-way ANOVA followed by Tukey’s post hoc test). b TIME endothelial cells (telomerase-immortalized human dermal microvascular endothelial cell line) were transfected with WT or K/R mutant Akt1 constructs and stimulated with LPS (5 μg/ml) for 0 and 6 h. Confocal imaging showed that expression of K/R mutant Akt1 prevents degradation of VE-cadherin. c WT mice were injected (i.v.) with liposome-encapsulated pmCherry-tagged WT or K/R mutant Akt1 constructs. Lungs harvested 96 h after injection were subjected to cryosection and stained with EC marker antibody vWF (green). Confocal imaging confirms expression of pmCherry-Akt1 (red) plasmid in lung endothelial cells. d-f Liposome-mediated delivery of Akt1 (WT) or K/R-mutated Akt1 in WT mice prevents degradation of VE-cadherin, mitigates LPS-induced lung vascular leak (EBA uptake), and reduces PMN transmigration (MPO assay). Shown are mean values ± SEM ( n = 3 or n = 5 mice/group; two-way ANOVA followed by Tukey’s post hoc test). g Model for E3 ligase CHFR regulation of endothelial junctional barrier integrity. Under baseline condition, constitutive Ang1-Tie2 signaling in EC maintains endothelial junctional barrier through Akt1 activation-mediated inhibition of the transcription factor FoxO1 activation and Ang-2 expression. During vascular inflammatory conditions such as sepsis, TLR4 signaling induces the expression of E3 ligase CHFR in a FoxO1-dependent manner. Then the upregulated CHFR mediates K 48 -linked polyubiquitylation and degradation of Akt1 and VE-cadherin (Tiruppathi et al., 2023) to disassemble EC junctional barrier. CHFR-mediated loss of FoxO1 negative regulator Akt1 expression in EC leads to increased FoxO1 expression which in turn promotes sustained expression of Ang-2 in EC to induce life-threatening pulmonary edema.

Article Snippet: Mouse mAb against Akt1 (catalog #sc-5298; IB, 1:1000; IP, 1 μg/100 μg cell lysate protein), and mouse mAb against FoxO1 (catalog #sc-374427; IS, 1:100) were from Santa Cruz Biotechnology.

Techniques: Transfection, Mutagenesis, Construct, Expressing, Imaging, Injection, Staining, Marker, Plasmid Preparation, Transmigration Assay, MPO Assay, Activation Assay, Inhibition

Journal: Nature Communications

Article Title: MRAP mediated adipocyte differentiation by thymic mesenchymal stromal cells contributes to thymic involution

doi: 10.1038/s41467-025-64973-z

Figure Lengend Snippet: A Representative immunofluorescence images of purified tMSCs showing anti-FoxO1 antibodies (green) counterstained with DAPI (nuclei, blue). Data are representative of 4 independent experiments. Scale bar, 50 µm. B RT-qPCR analysis of Mrap and FoxO1 expression following stimulated by a series of thymosin-α1 concentration to tMSCs ( n = 5 biological replicates/group). ***p < 0.005. C Western blot detected MRAP and phosphorylation of AKT and FoxO1 (p-AKT and p-FoxO1) or AKT and FoxO1 in tMSCs stimulated by various doses of thymosin-α1 for 14 days. The results were from three independent experiments. D Thymic MSCs were stimulated with or without 20 μg/mL of thymosin-α1 in the presence or absence of FoxO1 inhibitor (2 μM) for 14 days. Cell lysates were subjected to immunoblotting analysis to detect the MRAP and FABP4. The results were from three independent experiments. E Thymic MSCs were stimulated with 20 or 30 μg/mL of thymosin-α1 in the presence or absence of FoxO1 inhibitor (2 μM) under adipogenic differentiation culture for 21days. Oil red O staining showing the presence of lipid vesicles in adipocytic differentiated cells (two independent experiments). Scale bar, 100 µm. Data are presented as mean ± SD. p values were determined using one-way ANOVA with Tukey’s multiple-comparison test: ( B ). Source data are provided as a Source Data file.

Article Snippet: Afterward, the membranes were blocked with 5% (w/v) skim milk at room temperature and immunoblotted overnight at 4 ◦ C with rabbit polyclonal antibody against MRAP (Proteintech, #19512-1-AP), FABP4 (CST, #2120), pFoxO1 (CST, #9461), AKT (CST, #4691), p-AKT (CST, #13038), and mouse monoclonal antibody against FoxO1 (CST, #14952), α-tubulin (Proteintech, #66031-1-Ig) and GAPDH (Proteintech, #60004-1-Ig) according to the instruction, respectively.

Techniques: Immunofluorescence, Purification, Quantitative RT-PCR, Expressing, Concentration Assay, Western Blot, Phospho-proteomics, Staining, Comparison

Journal: Science Advances

Article Title: Atf3-mediated metabolic reprogramming in hepatic macrophage orchestrates metabolic dysfunction–associated steatohepatitis

doi: 10.1126/sciadv.ado3141

Figure Lengend Snippet: ( A ) Co-immunoprecipitation (Co-IP) was performed using hepatic macrophages isolated from C57BL/6J mice fed a chow diet. ( B ) Representative image of basal FoxO1/Atf3 dimerization in hepatic macrophages. ( C and D ) Hepatic macrophages were pretreated with 1 μM AS1842856 for 24 hours and infected with Lv-Gfp or Lv-Atf3 for 24 hours. The Pck2 activity (C) and glucose levels (D) were determined. ( E and L ) C57BL/6J mice were fed chow diet (CD) or WD for 8 or 16 weeks. The levels of acetyl-FoxO1 and Cd36 palmitoylation in hepatic macrophages were measured. ( F and M ) Lv-Atf3 , Atf3 M−/− , or control mice were fed WD for 20 weeks. The level of acetyl-FoxO1 and CD36 palmitoylation in liver macrophages was determined. ( G ) Hepatic macrophages were infected with Lv-Gfp or Lv-Atf3 for 36 hours. Protein samples were subjected to immunoprecipitation and Western blotting and developed with appropriate antibodies, as shown in the figures. ( H ) Co-IP was performed using hepatic macrophages isolated from C57BL/6J mice. ( I ) Hepatic macrophages were pretreated with 10 μM MS-275 for 24 hours and infected with Lv-Gfp or Lv-Atf3 for 24 hours. Protein samples were subjected to immunoprecipitation and Western blotting and developed with appropriate antibodies, as shown in the figures. ( J ) The FoxO1 has three putative Hdac1 deacetylating sites. ( K ) Human embryonic kidney (HEK) 293T cells cotransfected pCMV-Hdac1 or pCMV-Atf3 with pCMV-flag-FoxO1-Wt or pCMV-flag-FoxO1-Mut for 36 hours. Levels of acetylated FoxO1 were determined. ( N ) The levels of CD36 palmitoylation in Atf3 fl/fl or Atf3 M−/− macrophages were measured. ( O ) Hepatic macrophages were isolated from C57BL/6J mice, and CUT&Tag assay was performed. ( P ) The heatmap shows that palmitoylation-related genes were regulated by Atf3. ( Q and R ) Cd36 WT or Cd36 Mut macrophages were infected with Lv-Gfp or Lv-Atf3 for 24 hours. Fatty acid uptake and oxidation were measured. ( S ) Simplified model depicting the role of Atf3 in glucolipid metabolism. * P < 0.05; ** P < 0.01. NS, not significant.

Article Snippet: Antibodies against Atf3 (catalog no. ABS136180), Cd36 (catalog no. ABS127810), or Srebp1c (catalog no. ABS131802) were purchased from Absin Bioscience Inc. Antibodies against Cd68 (catalog no. PE-65187), Hdac1 (catalog no. 10197-1-AP), P-Acc1 (catalog no. 29119-1-AP), glucose-6-phosphatase (G6pc; catalog no. 66860-1-lg), phosphoenolpyruvate carboxykinase 1 (Pck1; catalog no. 16754-1-AP), Pck2 (catalog no. 14892-1-AP), stearoyl-CoA desaturase 1 (Scd1; catalog no. 28678-1-AP), Abhd17a (catalog no. 15854-1-AP), Zdhhc5) (catalog no. 21324-1-AP), transforming growth factor–β1 (Tgfβ1; catalog no. 21898-1-AP), or α-tubulin (catalog no. 11224-1-AP) were purchased from Proteintech Group Inc. Antibodies against FoxO1 (catalog no. 14952S), acetylated lysine (catalog no. 9441), fatty acid synthase (Fasn; catalog no. 3180S), Acc (catalog no. 3662S), AMPKα (catalog no. 2603), or P- AMPKα (catalog no. 2535) were purchased from Cell Signaling Technology.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Isolation, Infection, Activity Assay, Control, Western Blot

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 1. FoxO1 was predominantly expressed in ME cells. (A–D) ME cells were isolated from 8-week-old male Myh11- CreERT2/tdTomato (tdT)fl/fl mice (n = 4). Cre recombination was induced by tamoxifen injection 1 day before the assay. (A) Flow cytometry histograms. The Myh11-positive ME cells (tdT+) represented 10.6% (right) of salivary gland tissue cells without endothelial cells, hematocytes, and erythroid cells (CD31−C45−TER119−; 96.06%, left). (B,C) tdT fluorescence and FoxO1 immunofluorescence (IF) in FACS-sorted cells [crude, Myh(+), Myh(−)] (B). Bar = 20 μm. The cell population expressing FoxO1 and tdT double positive (%) in each FACS-sorted cells [crude, Myh(+), Myh(−)] (C). Threshold intensity was 30. *P < 0.05. (D) Expression of αSMA and FoxO1 in CD31−C45−TER119− cells (crude), tdT-positive [Myh(+)] and -negative [Myh(−)] cells. *P < 0.05. (E) tdT fluorescence and IF of FoxO1 and E-cadherin (E-cad) in submandibular glands (SMG) of Myh11-CreERT2/tdTfl/fl on embryonic day 16 (E16, n = 4) and at 8 weeks (8w, n = 3). The arrow head showed αSMA and FoxO1 double positive cells. Bar = 20 μm. All data were representative of three independent experiments. See also Supplementary Fig. S1.

Article Snippet: The cryosections (4 μm) were prepared and subjected to immunofluorescence using anti-mouse antibodies against FoxO1 (1:50; #2880; Cell Signaling Technology, Danvers, MA) and E-cad (1:100; #610181; BD Biosciences, Franklin Lakes, NJ), Eda (1:100; #PA5-72840; Invitrogen), Eda2r (1:100; #BS-7111R; Bioss), and phospho-NF-κB p65 (1:100; #3033; Cell signaling) overnight at 4 °C, followed by incubation with Alexa Fluor secondary antibody (1:200; Invitrogen) for 1 h at room temperature.

Techniques: Isolation, Injection, Flow Cytometry, Fluorescence, Immunofluorescence, Expressing

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 2. Overexpression of FoxO1 in ME cells. (A) Scatter plot of CD49f (x-axis) and EpCAM (y-axis). The cells were isolated from SMG in TP53 mutant female mice (n = 4) and analyzed by flow cytometry. EpCAMlowCD49fhigh-cells were sorted as ME cells (6.5%). (B) A schematic for integration of PiggyBac transposon vector plasmid. The Tet-On inducible gene expression system was used. FoxO1 expression was induced by doxycycline (Dox). (C) mCherry fluorescence merged with phase contrast in MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) for 48 h. (D) Expression of FoxO1 mRNA in MEPB-FoxO1 cells treated with and without Dox for 24 h. *P < 0.05. n = 3. (E) Immunoblotting for FoxO1, αSMA, Krt14, Krt5, and β-actin in MEPB-FoxO1 cells treated with and without Dox for 72 h. (F) FoxO1 luciferase assay in the presence of FoxO1 inhibitor (Inh.; AS1842856) at the indicated concentrations. pGL4 luciferase reporter vector (upper) was constructed to include three FoxO1-binding elements (daf16:TTGTTTA and mdaf16:TTGCTTA). FoxO1 transcriptional activity was measured. pRL-TK was used as internal control. The Renilla luciferase normalized the firefly luciferase. #P < 0.05 vs. control (Ctrl). *P < 0.05 vs. Dox. n = 5. (G) Expression of αSMA mRNA in ME cells treated with and without FoxO1 inhibitor (Inh.; AS1842856, 1 μM) for 72 h. *P < 0.05. n = 3. (H) Expression of FoxO1 and αSMA mRNA in siRNA-mediated knockdown of FoxO1 (siFoxO1) or control (si Ctrl) in ME cells. *P < 0.05. n = 3. (I) Immunoblotting for NF-κB/p65 and phospho-NF-κB/p65 in MEPB-FoxO1 cells treated with and without Dox at the indicated time-points. The signal intensity of phospho-NF-κB/p65 was normalized to that of NF-κB/p65 (ratio). All data were representative of three independent experiments. See also Supplementary Figs. S2 and S3.

Article Snippet: The cryosections (4 μm) were prepared and subjected to immunofluorescence using anti-mouse antibodies against FoxO1 (1:50; #2880; Cell Signaling Technology, Danvers, MA) and E-cad (1:100; #610181; BD Biosciences, Franklin Lakes, NJ), Eda (1:100; #PA5-72840; Invitrogen), Eda2r (1:100; #BS-7111R; Bioss), and phospho-NF-κB p65 (1:100; #3033; Cell signaling) overnight at 4 °C, followed by incubation with Alexa Fluor secondary antibody (1:200; Invitrogen) for 1 h at room temperature.

Techniques: Over Expression, Isolation, Mutagenesis, Flow Cytometry, Plasmid Preparation, Gene Expression, Expressing, Fluorescence, Western Blot, Luciferase, Construct, Binding Assay, Activity Assay, Control, Knockdown

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 3. Transcriptome profiling of FoxO1-expressing ME cells. The RNA samples were prepared from MEPB-FoxO1 cells treated with and without Dox for 72 h. (A) Heatmap of differentially expressed genes (control vs. FoxO1). (B) The number of up- and down-regulated genes based on fold change of comparison pair (FoxO1/ control ≥ 2, P < 0.05). (C–F) Enrichment of Gene Ontology terms for biological processes associated with up- (C) and down- (D) regulated genes. (E,F) Data from the gene set enriched analysis (GSEA). The top gene lists of normalized enrichment score (NES) are shown in (E). *P < 0.001. Enrichment plot of cyclin A B1 B2 associated events during G2 M transition are shown in (F). See also Supplementary Tables S2–S5.

Article Snippet: The cryosections (4 μm) were prepared and subjected to immunofluorescence using anti-mouse antibodies against FoxO1 (1:50; #2880; Cell Signaling Technology, Danvers, MA) and E-cad (1:100; #610181; BD Biosciences, Franklin Lakes, NJ), Eda (1:100; #PA5-72840; Invitrogen), Eda2r (1:100; #BS-7111R; Bioss), and phospho-NF-κB p65 (1:100; #3033; Cell signaling) overnight at 4 °C, followed by incubation with Alexa Fluor secondary antibody (1:200; Invitrogen) for 1 h at room temperature.

Techniques: Expressing, Control, Comparison

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 4. FoxO1 suppressed ME cell proliferation via cell cycle arrest. (A) Viability of MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) at the indicated time-points. (B,C) Cell proliferation rates were measured by BrdU incorporation assay. BrdU positive/DAPI (%, left) with and without Dox (2 µg/mL) for 24 h (B) or with and without transfection of siRNA for FoxO1 for 48 h (C). Immunofluorescent images were showed on the right (BrdU; green, DAPI; blue). (D–F) Expression of p27(KIP1) in MEPB-FoxO1 cells. Cells were treated with and without Dox (2 µg/mL) (D), pretreated with and without FoxO1 inhibitor (Inh.; AS1842856, 1 μM) (E) and transfected with siRNA for FoxO1 (F) in the presence of Dox (2 µg/mL) for 48h. The expression data of p21(CIP/WAF1) were shown in Fig. S4. (G) Chromatin immunoprecipitation-quantitative real-time PCR (ChIP-qPCR) analysis of the DNA binding activity of FoxO1 in ME cells. DNA sample was prepared from MEPB-FoxO1 cells treated with Dox (2 µg/mL) for 72 h. The associated DNA at the promoter regions of p21CIP/WAF1 (− 1722 to − 1712) and p27KIP1 (− 1036 to − 1026), after incubation with FoxO1 antibody-conjugated protein G beads, were immunoprecipitated and analyzed by qPCR. *P < 0.05. n = 3. All data were representative of three independent experiments. (H) A schematic for FoxO1-induced cell growth inhibition. See also Supplementary Fig. S4.

Article Snippet: The cryosections (4 μm) were prepared and subjected to immunofluorescence using anti-mouse antibodies against FoxO1 (1:50; #2880; Cell Signaling Technology, Danvers, MA) and E-cad (1:100; #610181; BD Biosciences, Franklin Lakes, NJ), Eda (1:100; #PA5-72840; Invitrogen), Eda2r (1:100; #BS-7111R; Bioss), and phospho-NF-κB p65 (1:100; #3033; Cell signaling) overnight at 4 °C, followed by incubation with Alexa Fluor secondary antibody (1:200; Invitrogen) for 1 h at room temperature.

Techniques: BrdU Incorporation Assay, Transfection, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Binding Assay, Activity Assay, Incubation, Immunoprecipitation, Inhibition

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 5. FoxO1 induced Eda/Eda2r expression in ME cells through NF-κB activation. (A,B) Gene expression (A) and immunoblotting (B) of Eda and Eda2r in MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) for 24 h. (C) Expression of Eda and Eda2r in Dox-treated (2 µg/mL, 72 h) MEPB-FoxO1 cells with and without FoxO1 inhibitor (Inh.; AS1842856, 10 μM) pretreatment for 24 h. (D) Expression of Eda and Eda2r in Dox-treated (2 µg/mL, 72 h) MEPB-FoxO1 cells with and without NF-κB inhibitor (MG132, 20 μM) pretreatment for 6 h. *P < 0.05. n = 3. All data were representative of three independent experiments. See also Supplementary Fig. S5.

Article Snippet: The cryosections (4 μm) were prepared and subjected to immunofluorescence using anti-mouse antibodies against FoxO1 (1:50; #2880; Cell Signaling Technology, Danvers, MA) and E-cad (1:100; #610181; BD Biosciences, Franklin Lakes, NJ), Eda (1:100; #PA5-72840; Invitrogen), Eda2r (1:100; #BS-7111R; Bioss), and phospho-NF-κB p65 (1:100; #3033; Cell signaling) overnight at 4 °C, followed by incubation with Alexa Fluor secondary antibody (1:200; Invitrogen) for 1 h at room temperature.

Techniques: Expressing, Activation Assay, Gene Expression, Western Blot

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 6. Inhibition of FoxO1 inhibited development of the primitive epithelium of SMG ex vivo. Epithelia of SMG rudiments on E14.5 (n = 4), were mounted in Matrigel drops and cultured in the presence of FGF1 and FGF7 with and without FoxO1 inhibitor (Inh.; AS1842856, 10 μM) for 3 days. (A) Phase contrast images. Bar = 500 μm. (B) The image of immnofluorescent of αSMA, Eda, Eda2r, and phospho-NF-κB after 3 days of culture. Nuclei were stained with DAPI. Bar = 20 μm. (C) Expression of Eda and Eda2r after 3 days of culture. *P < 0.05. All data were representative of three independent experiments. See also Supplementary Figs. S6 and S7.

Article Snippet: The cryosections (4 μm) were prepared and subjected to immunofluorescence using anti-mouse antibodies against FoxO1 (1:50; #2880; Cell Signaling Technology, Danvers, MA) and E-cad (1:100; #610181; BD Biosciences, Franklin Lakes, NJ), Eda (1:100; #PA5-72840; Invitrogen), Eda2r (1:100; #BS-7111R; Bioss), and phospho-NF-κB p65 (1:100; #3033; Cell signaling) overnight at 4 °C, followed by incubation with Alexa Fluor secondary antibody (1:200; Invitrogen) for 1 h at room temperature.

Techniques: Inhibition, Ex Vivo, Cell Culture, Staining, Expressing