anti foxo1 mab  (Cell Signaling Technology Inc)

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