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 β-actin (catalog #A5441; IB, 1:2000) was from Sigma-Aldrich Inc. Rabbit pAb against GFP (catalog #50430-2-AP; IB, 1:2000), rabbit pAb against CHFR (catalog #12169-1-AP; IB, 1:1000), rabbit pAb against Angiopoietin-2 (catalog #24613-1-AP; IB, 1:1000), rabbit pAb against β-catenin (catalog #61067-2-AP; IB, 1:1000), and rabbit pAb against mCherry (catalog #26765-1-AP; IB, 1:1000) were from Proteintech.

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 β-actin (catalog #A5441; IB, 1:2000) was from Sigma-Aldrich Inc. Rabbit pAb against GFP (catalog #50430-2-AP; IB, 1:2000), rabbit pAb against CHFR (catalog #12169-1-AP; IB, 1:1000), rabbit pAb against Angiopoietin-2 (catalog #24613-1-AP; IB, 1:1000), rabbit pAb against β-catenin (catalog #61067-2-AP; IB, 1:1000), and rabbit pAb against mCherry (catalog #26765-1-AP; IB, 1:1000) were from Proteintech.

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

Journal: Cells

Article Title: Host SNARE Proteins Mediate Lysosome and PVM Fusion to Support Plasmodium Liver Infection

doi: 10.3390/cells15070584

Figure Lengend Snippet: Localization of host SNARE proteins at the Plasmodium PVM during liver-stage infection. ( A – C ) Confocal live cell imaging of GFP-tagged SNARE proteins VAMP7-GFP ( A ), VAMP8-GFP ( B ), and Stx7-GFP ( C ) in P. berghei -infected HeLa cells expressing mCherry (parasite cytoplasm, red) at 6, 24, and 48 hpi. SNARE localization is shown in green. ( D – G ) Immunofluorescence analysis of Vti1B ( D ), VAMP7 ( E ), VAMP8 ( F ), and Stx7 ( G ) in HeLa cells fixed at 0.5, 1, and 1.5 hpi. SNAREs were detected with anti-GFP (green) or anti-Vti1B (green); the PVM was labeled with anti-UIS4 (red); and DAPI (blue) stained nuclei. ( H ) Localization of SNARE proteins (anti-Vti1B/anti-GFP in green) with the PVM marker UIS4 (red) at 24 hpi, visualized by expansion microscopy (5-fold expanded). Merged channels (yellow) highlight PVM-SNARE protein association. Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm ( A – G ); 10 μm ( H ).

Article Snippet: The cells were then incubated in 10% FCS/PBS for 1 h, at room temperature with primary antibodies: Vti1B (mouse mAb 1:1000 (1:500 PS-ExM), BD Transduction Laboratories #611404, Allschwil, Switzerland), UIS4 (rabbit 1:1000, P. sinnis ; chicken 1:10000, Proteogenix, Schiltigheim, France), hLAMP1 (mouse mAb 1:1000 (1:500 PS-ExM), DSHB H4A3 (Iowa City, USA); rabbit pAb 1:1000, Cell Signaling #9091, Allschwill, Switzerland), GFP (rabbit pAb 1:1000, Origene TA100030, Herford, Germany; mouse mAb 1:1000, Roche AQ160, Basel, Switzerland), and α-tubulin (guinea pig pAb 1:500, ABCD AA345, Geneva, Switzerland).

Techniques: Infection, Live Cell Imaging, Expressing, Immunofluorescence, Labeling, Staining, Marker, Microscopy