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: Nature Microbiology

Article Title: Specialized RNA decay fine-tunes monogenic antigen expression in Trypanosoma brucei

doi: 10.1038/s41564-026-02289-4

Figure Lengend Snippet: a , b , Fluorescence microscopy analysis of ESB2 12myc localization following ESB3 and ESAP1 RNAi ( a , b ), 6myc ESB3 localization following ESB2 and ESAP1 RNAi ( b ) and 6myc ESAP1 localization following ESB2 and ESB3 RNAi ( b ). c , ESB2, ESB3 and ESAP1 protein levels following VEX2, ESB2, ESB3 and ESAP1 RNAi. The values were derived from protein blotting analysis of three biological replicates per cell line and are represented in a floating bar graph in which the box spans between minimum and maximum values, the centre line is the median, and all datapoints are depicted. Two-tailed unpaired t -tests were applied; NS, non-significant; ** P < 0.01. d – i , Fluorescence microscopy analysis of Pol-I localization following VEX2, ESB1, ESB2, ESB3 and ESAP1 knockdown ( d , e ); ESB1 12myc , ESB2 12myc , 6myc ESB3 and 6myc ESAP1 localization following VEX2 knockdown ( f , g ); 6myc VEX2 localization following ESB2, ESB3 and ESAP1 knockdown ( h , i ). The graphs in b , e , g and i depict mean values of two biological replicates; >100 G1 cells per condition were analysed; all analyses were performed at 24 h post-induction. j , Heatmap summarizing the microscopy analyses; C1, clone 1; C2, clone 2. k , Diagram depicting the complex network of co-dependencies at the ESB and interface with the SLAB. The direction of the arrows indicates that the protein upstream is required for the localization of the protein downstream to its corresponding nuclear compartment. l , Fluorescence microscopy colocalization analysis of GFP-myc ESB2 and 6HA ESB3. The images in a , d (left), h and l were acquired using a Zeiss AxioObserver, whereas the images in d (right) and f were acquired using a Zeiss LSM980 Airyscan 2. All correspond to 3D projections by brightest intensity of 0.1-μm stacks. DNA was stained with DAPI (cyan or grey). Scale bars, 2 or 5 μm. m , Immunoprecipitation of GFP-myc ESB2 using GFP nanobody-coated beads followed by protein-blot analysis using an anti-HA antibody to detect the prey ( 6HA ESB3) and an anti-GFP antibody to detect the bait ( GFP-myc ESB2). IP, immunoprecipitation. The protein blots are representative of four independent experiments; a 6HA ESB3 single tagged cell line was used as a negative control. n , Violin plots depicting a comparative transcriptomic analysis between VEX2 ( n = 3), ESB2 ( n = 5), ESB3 ( n = 3) and ESAP1 ( n = 3) RNAi cell lines for ‘active’ ESAGs ; n values correspond to biological replicates. log 2 (FC), fold change in transcript abundance between RNAi (24 h post-induction) and the parental cell line. The violins span between minimum and maximum values, centre lines correspond to the mean and all datapoints are shown. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparison test; **** P < 0.0001. Diagram in m created in BioRender; Faria, J. https://BioRender.com/oia6hdu (2026).

Article Snippet: The following primary antibodies were used: mouse α-myc (Millipore, clone 4A6, 1:10,000), mouse anti-Ty (BB2, Invitrogen, 1:5,000), mouse monoclonal anti-HA (Sigma, clone HA-7, 1:2,000), rabbit polyclonal anti-GFP (LifeTech, 1:2,000) and mouse α-EF1α (Millipore, clone CBP-KK1, 1:30,000).

Techniques: Fluorescence, Microscopy, Derivative Assay, Two Tailed Test, Knockdown, Staining, Immunoprecipitation, Negative Control, Comparison