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 scrambled-siRNA (sc-siRNA) or CHFR-siRNA. At 72 h post-transfection, cells stimulated with rAng1 (400 ng/ml) for different time intervals were used for IB analysis to determine phosphorylation of Akt1 at S473 and T308 ( n = 3 independent experiments). b Control HMEC and CHFR knockdown HMEC stimulated with rAng1 as above were stained with phospho-473-Akt1 specific antibody. c Control HMEC and CHFR knockdown HMEC were transfected with the Akt1 biosensor plasmid and then stimulated with rAng1 as above to assess live cell Akt1 activity by measuring the FRET ratio ( p <0.0001) ( left panel shows basal Akt activity; right panel shows Akt activity in response to rAng1 challenge). d-f Control HMEC and CHFR-siRNA treated HMEC stimulated with rAng1 were used for IB analysis to determine tyrosine phosphorylation Tie2 ( d ), phosphorylation of GSK3β at S9 and phosphorylation of β-catenin ( e ), ( n = 3 independent experiments). a, d-e Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Rabbit polyclonal antibody (pAb) against phospho-Tie2 (catalog #AF3909; IB, 1:1000), goat polyclonal antibody (pAb) against PDGFRβ (catalog #AF1042; IS, 1:100) were from R&D system.

Techniques: Transfection, Phospho-proteomics, Control, Knockdown, Staining, Plasmid Preparation, Activity Assay

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: Rabbit polyclonal antibody (pAb) against phospho-Tie2 (catalog #AF3909; IB, 1:1000), goat polyclonal antibody (pAb) against PDGFRβ (catalog #AF1042; IS, 1:100) were from R&D system.

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

Journal: bioRxiv

Article Title: Dual targeting of astrocytic and endothelial GLUT1 enables functional rescue in GLUT1 deficiency syndrome

doi: 10.64898/2026.03.04.709430

Figure Lengend Snippet: (A–C) Behavioral analyses in six groups: wild-type, systemic Glut1 del/+ mice, Aldh1l1-Cre (Al cre); Glut1 +/+ mice, Al cre; Glut1 flox/+ mice, Tie2-Cre; Glut1 +/+ mice, and Tie2-Cre; Glut1 flox/+ mice (n = 6–15 mice per group). (A) Object location test (OLT) and (B) novel object recognition test (NORT) were used to assess spatial and recognition memory (discrimination index). (C) The foot-slip test evaluated motor coordination (missed steps per 50 steps). (D) CSF-to-blood glucose ratio measured in the same cohorts (n = 5–6 mice per group). Blood glucose levels were comparable across groups (see ). (E) Schematic of the in vivo recording setup for thalamic interstitial fluid (ISF) glucose measurement. Illustration created with BioRender. (F) ISF glucose measured following oral glucose administration (0.03 g per mouse, gavage). The increase in ISF glucose was markedly blunted in systemic, astrocyte-specific, and endothelial-specific Glut1 haploinsufficient mice compared with wild-type controls (n = 4–5 mice per group). Data are presented as mean ± SEM; dots represent individual mice. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple-comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Mice harboring the Tie2-Cre (stock no. #004128), Aldh1l1-Cre (stock no. #023748), Aldh1l1-CreERT2 (stock no. #029655) drivers, tdTomato-floxed STOP reporter mice (tdTomato reporter mice) (stock no. 007914) and floxed Glut1 mice (stock no. #031871) were all obtained from the Jackson Laboratory, backcrossed over 10 generations to the C57BL/6J genetic strain background.

Techniques: In Vivo, Comparison