primary antibody against p62  (Cell Signaling Technology Inc)

Journal: Journal of Lipid Research

Article Title: The compound LY295427 antagonizes 25-hydroxycholesterol through binding to INSIG

doi: 10.1016/j.jlr.2026.101015

Figure Lengend Snippet: LY295427 binds to INSIG. A: The structure of the LY295427 probe and a schematic illustration of the detection procedure for LY295427 -binding proteins. B: CHO cells were incubated in cholesterol depletion medium supplemented with or without 20 μM LY295427 , 20 μM LY295427 probe, and 1 μg/ml 25-HC for 24 h. The cells were harvested and subjected to immunoblotting. C–G: CHO cells were transfected with pCMV-INSIG-1-Myc, pCMV-INSIG-2-Myc, or pCMV-INSIG-2 (F115A)-Myc plasmid for 24 h and incubated in cholesterol depletion medium supplemented with or without LY295427 probe, LY295427 , and 25-HC for 24 h. The cells were harvested and treated as described in A. Input and pellet were immunoblotted with anti-Myc antibody. H: HEK293T cells were transfected with pLVX-SCAP-EGFP or pLVX-SCAP(D443N)-EGFP plasmid for 24 h and incubated in cholesterol depletion medium supplemented with or without LY295427 probe and cholesterol/CDX for 24 h. The cells were harvested and treated as described in A. Input and pellet were immunoblotted with anti-Myc and anti-SCAP (9D5) antibodies. I: The model diagram illustrating the action of 25-HC and LY295427 .

Article Snippet: The primary antibodies used were as follows: mouse monoclonal antibody against β-actin (#A5441; Sigma), mouse monoclonal antibody against FLAG-tag (#F3165; Sigma), mouse monoclonal antibody against T7-tag (#69522; Merck), mouse monoclonal antibody against HA-tag (#H3663; Sigma), mouse monoclonal antibody P4D1 against ubiquitin (#SC-8017; Santa Cruz Biotechnology), mouse monoclonal antibody 9D5 against SCAP (#CRL-2347; ATCC), mouse monoclonal antibody 7D4 against SREBP2 (#CRL-2198; ATCC), anti-farnesyl-diphosphate farnesyltransferase 1 (FDFT1) antibody (#13128; Proteintech), anti-lanosterol synthase (LSS) antibody (#18693; Proteintech), mouse monoclonal antibody 9E10 against MYC-tag, mouse monoclonal antibody A9 against HMGCR, mouse monoclonal antibody 1D2 against SREBP2, rabbit polyclonal antibody against enhanced GFP (EGFP)-tag, rabbit polyclonal antibody against LDLR, and rabbit polyclonal antibody against HMGCR, which were prepared in our laboratory.

Techniques: Binding Assay, Incubation, Western Blot, Transfection, Plasmid Preparation

Journal: Neoplasia (New York, N.Y.)

Article Title: BPGM as an intrinsic brake to constrain metastasis through phospho-epigenetic-mediated carnitine biosynthesis suppression

doi: 10.1016/j.neo.2026.101299

Figure Lengend Snippet: Overexpression of BPGM inhibits tumor metastasis in vitro and in vivo . (A) Silencing BPGM promoted migration of tumor cells. Tumor cells stably expressing shBPGM and its control cells (shCtrl) were examined. Scale bar, 200 μm. (B) Overexpressing BPGM suppressed migration of tumor cells. Tumor cells stably expressing BPGM and its control cells (Ctrl) were examined. Scale bar, 200 μm. (C) The mRNA levels of MMP2 and MMP9 reduced in BPGM-overexpressing tumor cells. SK-HEP-1 cells stably expressing BPGM (BPGM-OE) and its ctrl cells were employed to detect the mRNA levels of MMP2 and MMP9 by qPCR analysis. β-actin was used as an internal control. (D-E) Xenografts of stable Bpgm-overexpressing cells displayed a lower rate of liver and lung metastasis and less metastatic nodules in the liver. For (D), Hepa-Ctrl ( n = 7) and Hepa-BPGM sublines ( n = 6) were inoculated under the capsule of the left hepatic lobe of C57BL/6 mice. Upper panel, a schematic diagram of orthotopic hepatic implantation model. The number of metastatic rate and nodules is shown (D, lower panel). Scale bar, 1 cm. Hematoxylin-eosin staining was performed on serial sections of livers (E, left panel) and lungs (E, right panel) to detect the metastatic nodules. The red arrows indicated the metastatic nodules. Scale bar in left panel, 200 µm; Scale bar in right panel, 100 μm. (F-H) Overexpressing of BPGM inhibited lung metastasis of tumor xenografts. Scale bar in F, 1 cm. B16-F10 cells transfected with Ctrl ( n = 6) or BPGM-OE ( n = 6) was injected into the tail vein of C57BL/6 mice, respectively. Upper panel in F, a schematic diagram of lung metastasis model by tail vein injection. H&E staining of lung sections was performed to observe metastatic foci (G). For G, scale bar in left panel, 500 μm; scale bar in right panel, 200 μm. The number of melanoma nodules and lung metastasis is shown in H. (I) The model deciphers the inhibitory role of BPGM in tumor metastasis. Error bar: mean ± SEM. P -values are labeled above the bar chart.

Article Snippet: The antibodies used included mouse antibody against β-actin (BM0627, Boster, Wuhan, China), rabbit antibody against BPGM (17173-1-AP, Proteintech), EZH2 (F0281, Selleck), phospho-EZH2 (Thr345) (TA3584S, Abmart, Shanghai, China), phospho-CDK1 (Thr14) (AP1465, Abclonal, Wuhan, China), ubiquitin (10201-2-AP, Proteintech), HIF1α (36169, Cell Signaling Technology, CST, Beverly, MA, USA), H3K4me3 (91264, Active Motif), H3K79me3 (cat 49-1020, Thermos Fisher), H3K9me3 (61014, Active Motif), H3K27me3 (91168, Active Motif) and Histone 3 (F0057, Selleck).

Techniques: Over Expression, In Vitro, In Vivo, Migration, Stable Transfection, Expressing, Control, Staining, Transfection, Injection, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The Hsp40 cochaperone DNAJC7 regulates polyglutamine aggregation and exhibits context-dependent effects on polyglycine aggregation

doi: 10.1016/j.jbc.2026.111292

Figure Lengend Snippet: An inducible cell-based model of nuclear, detergent-resistant, p62-positive polyglutamine (polyQ) protein aggregates monitored by an FRET-based reporter. A , schematic of lentiviral constructs for doxycycline-inducible expression of nuclear-localized fluorescent proteins fused to a C-terminal ataxin-3 with 79 glutamines. HEK293T cells engineered with CRISPRi machinery were transduced with these constructs, and clones expressing both fluorescent proteins were selected to generate the NLS-FRET-Q79 cell line. B , fluorescence imaging of NLS-FRET-Q79 cells at 1 day and 5 days of doxycycline. C , immunofluorescence staining for endogenous p62 in NLS-FRET-Q79 cells after 5 days of doxycycline. D , fluorescence imaging of NLS-FRET-Q79 cells at 5 days of doxycycline before and after treatment with detergent, in the same field of view. E , schematic illustrating how polyQ aggregation gives rise to a “FRET-high” population observable by flow cytometry. F , flow cytometry plots at 1 day or 5 days of doxycycline, before and after treatment with detergent. All scale bars represent 10 μm. CRISPRi, CRISPR interference; HEK293T, human embryonic kidney 293T cell line; NLS, nuclear localization signal.

Article Snippet: Primary antibody against p62 (clone D5L7G, Cell Signaling, 88588) was diluted 1:1000 in blocking solution and incubated overnight at 4 °C.

Techniques: Construct, Expressing, Transduction, Clone Assay, Fluorescence, Imaging, Immunofluorescence, Staining, Flow Cytometry, CRISPR

Journal: The Journal of Biological Chemistry

Article Title: The Hsp40 cochaperone DNAJC7 regulates polyglutamine aggregation and exhibits context-dependent effects on polyglycine aggregation

doi: 10.1016/j.jbc.2026.111292

Figure Lengend Snippet: An inducible model of polyglycine (polyG) aggregation reveals detergent-resistant, p62-positive nuclear inclusions, and seeding activity. A , schematic of lentiviral constructs for generating the NLS-FRET-G100 cell line expressing the upstream ORF (uORF) of the NOTCH2NLC gene with a polyG tract of 100 residues. B , immunofluorescence staining for p62 of NLS-FRET-G100 cells at 5 days of doxycycline. C , fluorescence imaging of NLS-FRET-G100 cells at 5 days of doxycycline before and after detergent treatment, in the same field of view. D , flow cytometry plots at 1 and 5 days of doxycycline treatment and the latter before and after detergent treatment. E , flow cytometry plots of NLS-FRET-G100 cells at 4 days of doxycycline with or without proteasomal inhibitor (carfilzomib). All scale bars represent 10 μm. NLS, nuclear localization signal.

Article Snippet: Primary antibody against p62 (clone D5L7G, Cell Signaling, 88588) was diluted 1:1000 in blocking solution and incubated overnight at 4 °C.

Techniques: Activity Assay, Construct, Expressing, Immunofluorescence, Staining, Fluorescence, Imaging, Flow Cytometry

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: 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 Lung tissues from Chfr fl/fl and Chfr ΔEC mice were collected at 0 and 6 h after i.p. injection of LPS (10 mg/kg bodyweight). Lung sections were stained with antibodies specific to vWF (EC marker, green) and Akt1 (red). b Lung tissues from Chfr fl/fl and Chfr ΔEC mice were collected at 0 and 6 h after i.p. injection of LPS (10 mg/kg bodyweight). Lung sections were stained with antibodies specific to PECAM1 (EC marker, red) and PDGFRβ (pericyte marker, green). c Lung tissues from Chfr fl/fl and Chfr ΔEC mice were used for RT-qPCR to measure mRNA expression of Ang1 and Tie-2.

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: Injection, Staining, Marker, Quantitative RT-PCR, 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 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: 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, 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 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-b CHFR deficiency in EC prevents LPS-induced ubiquitylation of Akt1. HLMVEC transfected with sc-siRNA or CHFR-siRNA. At 72 h post-transfection, cells were challenged with LPS (5 μg/ml) for 6 h in the presence of the proteasomal inhibitor MG132 (10 μM). Cell lysates were immunoprecipitated with anti-Akt1 mAb and blotted with antibodies specific to K 48 -linked poly-Ub or K 63 -linked poly-Ub ( n = 2 independent experiments) ( a ). Chfr fl/fl (WT) and Chfr ΔEC mice were challenged with LPS (10 mg/kg; i.p.) for 6 h. After the LPS challenge, lungs harvested were used to determine ubiquitylation of Akt1 as above in a (b) . c-f CHFR induces ubiquitylation of activated Akt1. c Control and CHFR-depleted HLMVEC were pretreated with inhibitors of PDK1 and mTORC2 for 30 min and then exposed to LPS (5 μg/ml). Thereafter, cell lysates were used for IB to assess phosphorylation of Akt1. d-e HLMVEC pretreated with or without PDK1 and mTORC2 inhibitors for 1 h and stimulated with LPS (6 h) in the presence of MG123 (10 μM) showed that CHFR binds and ubiquitylates phosphorylated Akt1. f HEK-293T cells were transfected with HA-tagged ubiquitin (HA-Ub) (0.5 μg/ml) alone or co-transfected with N-terminal GFP-tagged WT-CHFR (1.5 μg/ml), N-terminal pmCherry-tagged WT-Akt1 (1.5 μg/ml), and phosphorylation-defective Akt1 mutant (Akt1T308A/S473A) (1.5 μg/ml) plasmids. Thirty-six hours after transfection, cells were incubated with MG123 (10 μM) for 3h, and cell lysates were used to determine phosphorylation-dependent ubiquitylation of Akt1.

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, Immunoprecipitation, Control, Phospho-proteomics, Ubiquitin Proteomics, Mutagenesis, Incubation

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 Schematics of the domain structures of human CHFR WT and mutants lacking forkhead-associated domain (ΔFHA-CHFR), RING finger domain (ΔRF-CHFR), cysteine-rich domain (ΔCR-CHFR), or poly-ADP ribose binding zinc-finger domain (ΔPBZ-CHFR) used in experiments. b Immunoblot showing expression of eGFP-tagged CHFR (WT) and CHFR mutants (1.5 μg/ml), along with pmCherry-tagged WT-Akt1 (1.5 μg/ml) in HEK-293T cells. c Transfected HEK-293T cells were used for anti-GFP agarose beads pull-down assays. Results show that WT-CHFR and CHFR mutants bind to WT-Akt1 in vitro . Bottom panel shows quantification of CHFR binding to Akt1 as a ratio of Akt1-to-GFP-CHFR. arb. units, arbitrary units. d HEK-293T cells transfected with WT-HA-Ub (0.5 μg/ml) alone or co-transfected with WT-CHFR (1.5 μg/ml), ΔFHA-CHFR (1.5 μg/ml), ΔRF-CHFR (1.5 μg/ml), and WT-Akt1 (1.5 μg/ml) were used to assess ubiquitylation of Akt1. At 36 h after transfection, cells were incubated with MG132 (10 μM) for 3 h, and then cell lysates were used for IB analysis. e-f HEK-293T cells transfected with WT-HA-Ub (0.5 μg/ml) or HA-tagged Ubiquitin where all Lysin (K) residues were mutated to Alanine (A) except at K48 or K63, along with WT-CHFR, and WT-Akt1 were used to determine CHFR-mediated linkage specific polyubiquitylation of Akt1.

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: Binding Assay, Western Blot, Expressing, Transfection, In Vitro, Incubation, Ubiquitin Proteomics

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: HEK-293T cells were transfected with pmCherry-Akt1 (WT), eGFP-CHFR (WT), and HA-Ub (WT). The transfected cells were stained with K 48 -linkage specific antibody. Confocal imaging showing the co-localization of HA-Ub (WT), pmCherry-Akt1 (WT), and eGFP-CHFR (WT).

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, 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 schematic showing the workflow for LC-MS analysis. HEK293 cells were transfected with pmCherry-Akt1 (WT), eGFP-CHFR (WT), and HA-Ub (WT). At 48 h post-transfection, cells were collected and subjected to urea lysis followed by trypsin digestion. The digested peptides were affinity-purified and immunoprecipitated with anti-di-glycine remnant antibodies to enrich for ubiquitylated peptides, followed by LC-MS/MS analysis. b shows the Akt1 ubiquitylated “K” containing peptides identified by LC-MS. c Model of Akt1 protein structure showing ubiquitylation sites. d HEK-293T cells transfected with WT-HA-Ub (0.5 μg/ml) alone or co-transfected with WT-CHFR (1.5 μg/ml), WT-Akt1 (1.5 μg/ml), or Ub-defective Akt1 (K30R, K268R, K39R+K268R, and K30R+K39R+K154R+268R) plasmids were used to study ubiquitylation of Akt1. IB analysis showed that the CHFR-induced ubiquitylation of Akt1 was blocked in “K to R” Akt1 mutants expressing cells compared with WT-Akt1.

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: Liquid Chromatography with Mass Spectroscopy, Transfection, Lysis, Affinity Purification, Immunoprecipitation, 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