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: Scrambled-siRNA (Sc-siRNA) and human ( h )-specific siRNA1 against CHFR (A pool of 3 target-specific siRNAs, sense: 5′-CUCUGUGGCAAGUGAUGAAtt-3′; sense: 5′-GAAGAAGAUGUGCAAAGUAtt; sense: 5′-GGUAUAGUAUGGUAUUUGAtt-3′) were obtained from Santa Cruz Biotechnology. pEGFP-C2 plasmid (Addgene catalog #61853) encoding wild-type human CHFR (WT h -CHFR), h CHFR mutant lacking forkhead-associated domain (ΔFHA-CHFR), h CHFR mutant lacking RING finger domain (ΔRF-CHFR), mutant lacking cysteine-rich domain (ΔCR-CHFR), and mutant lacking poly-ADP ribose binding zinc-finger domain (ΔPBZ-CHFR) were custom prepared by Genscript (Piscataway, NJ). pmcherry-C1 plasmid (Addgene catalog #86631) encoding wild-type human Akt1 (WT h -Akt1), phopsho-defective h Akt1 (T308A and S473A) mutant and ubiquitylation-defective h Akt1 (K30R, K39R, K39R+154R and K30R+K39R+K154R+K268R) mutants were custom prepared by Genscript (Piscataway, NJ).

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: Scrambled-siRNA (Sc-siRNA) and human ( h )-specific siRNA1 against CHFR (A pool of 3 target-specific siRNAs, sense: 5′-CUCUGUGGCAAGUGAUGAAtt-3′; sense: 5′-GAAGAAGAUGUGCAAAGUAtt; sense: 5′-GGUAUAGUAUGGUAUUUGAtt-3′) were obtained from Santa Cruz Biotechnology. pEGFP-C2 plasmid (Addgene catalog #61853) encoding wild-type human CHFR (WT h -CHFR), h CHFR mutant lacking forkhead-associated domain (ΔFHA-CHFR), h CHFR mutant lacking RING finger domain (ΔRF-CHFR), mutant lacking cysteine-rich domain (ΔCR-CHFR), and mutant lacking poly-ADP ribose binding zinc-finger domain (ΔPBZ-CHFR) were custom prepared by Genscript (Piscataway, NJ). pmcherry-C1 plasmid (Addgene catalog #86631) encoding wild-type human Akt1 (WT h -Akt1), phopsho-defective h Akt1 (T308A and S473A) mutant and ubiquitylation-defective h Akt1 (K30R, K39R, K39R+154R and K30R+K39R+K154R+K268R) mutants were custom prepared by Genscript (Piscataway, NJ).

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-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: Scrambled-siRNA (Sc-siRNA) and human ( h )-specific siRNA1 against CHFR (A pool of 3 target-specific siRNAs, sense: 5′-CUCUGUGGCAAGUGAUGAAtt-3′; sense: 5′-GAAGAAGAUGUGCAAAGUAtt; sense: 5′-GGUAUAGUAUGGUAUUUGAtt-3′) were obtained from Santa Cruz Biotechnology. pEGFP-C2 plasmid (Addgene catalog #61853) encoding wild-type human CHFR (WT h -CHFR), h CHFR mutant lacking forkhead-associated domain (ΔFHA-CHFR), h CHFR mutant lacking RING finger domain (ΔRF-CHFR), mutant lacking cysteine-rich domain (ΔCR-CHFR), and mutant lacking poly-ADP ribose binding zinc-finger domain (ΔPBZ-CHFR) were custom prepared by Genscript (Piscataway, NJ). pmcherry-C1 plasmid (Addgene catalog #86631) encoding wild-type human Akt1 (WT h -Akt1), phopsho-defective h Akt1 (T308A and S473A) mutant and ubiquitylation-defective h Akt1 (K30R, K39R, K39R+154R and K30R+K39R+K154R+K268R) mutants were custom prepared by Genscript (Piscataway, NJ).

Techniques: Transfection, Immunoprecipitation, Control, Phospho-proteomics, Ubiquitin Proteomics, Mutagenesis, Incubation

Journal: Cell Death Discovery

Article Title: Inhibiting HSP27 activates the XBP1s/CerS1 interplay, which triggers DRP1-driven mitophagy, thereby protecting against cell death and promoting the KSHV lytic cycle in primary effusion lymphoma cells

doi: 10.1038/s41420-026-02979-2

Figure Lengend Snippet: BC3 and BCBL1 cell lines were treated or not with 10 and 20 μM J2 (HSP27 inhibitor). After 24 h ( A ) the cytotoxic effect of J2 was measured by trypan blue assay. The histograms represent the percentage of cell viability relative to the control. Data are shown as the mean plus S.D. of three experiments. p value * <0.05, as calculated by ANOVA test. B BiP and CHOP expression as evaluated by western blot analysis. Histone H3 (H3) was used as loading control. The histograms represent the densitometric analysis of the ratio of BiP/H3 and CHOP/H3. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by ANOVA test. BC3 and BCBL1 cell lines treated or not with J2 (20 μM) for 4 h were analyzed by western blot for the expression of XBP1s ( C ) and after 24 h of treatment for cleaved PARP (clPARP) ( D ). GAPDH was used as loading control. The histograms represent the densitometric analysis of the ratio of XBP1s /GAPDH and clPARP/GAPDH. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by Student’s t -test. BC3 and BCBL1 cell lines were knocked down for HSP27 (siHSP27) or scramble treated, using siRNA from different companies (Santa Cruz and Dharmacon respectively), for 24 h and the expression of BiP ( E ) and XBP1s was evaluated by western blot analysis while the effect on cell viability was evaluated by trypan blue assay ( F ). Histone H3 (H3) was used as loading control. The histograms represent the densitometric analysis of the ratio of HSP27/H3, BiP/H3 and XBP1s/H3. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by Student’s t -test.

Article Snippet: For transfection 10 μl of INTERFERin reagent (Polyplus, Illkirch, France; cat. n. 101000016) in combination with 50 pmol of specific small interfering RNA (siRNA duplex) or with non-targeting (scramble) siRNA (Santa Cruz Biotechnology, Inc., Heidelberg, Germany cat. n. sc-29350) were diluted in 200 μl RPMI without serum and antibiotics, incubated for 10 minutes at RT then added to each well.

Techniques: Control, Expressing, Western Blot

Journal: Nature Communications

Article Title: Mycobacterium tuberculosis modulates phosphorylation of host ATP6V1E1 to promote intracellular survival

doi: 10.1038/s41467-026-69331-1

Figure Lengend Snippet: a Quantification of western blots to screen Kinase library regulated ATP6V1E1 phosphorylation. b In vitro phosphorylation assay of ATP6V1E1. The proteins of GFP-BMX, Flag-ATP6V1E1 and HA-ATP6V1E1 Y56/Y57A were overexpressed and purified in HEK293T cells. c–e Detection of ATP6V1E1 phosphorylation in macrophages transfected with Bmx siRNA ( c ), treated with BMX inhibitor (10 nM) ( d ) or in Bmx +/- macrophages ( e ), then infected with H37Rv (MOI = 5) for indicated times. ratio: p-ATP6V1E1 Y56/57 /ATP6V1E1. f Subcellular fractionation of macrophages from WT or Bmx +/- mice or WT macrophages under the treatment with BMX-IN (10 nM) for 1 h. Cytosolic (C) and membrane (M) fractions were immunoblotted with antibodies against the V1 domain subunit ATP6V1E1 and the V0 domain subunit ATP6V0d1. α-Tubulin and V0d1 serve as loading controls for cytosolic and membrane fractions, respectively. g Representative LysoTracker staining of macrophages transfected with scrambled or Bmx -specific siRNA and then infected with H37Rv (MOI = 5) for the indicated times. Scale bar, 10 μm. h Quantification of LysoTracker fluorescence intensity (mean ± SEM). i Intracellular CFU in macrophages transfected scrambled or Bmx -specific siRNA and then infected with H37Rv (MOI = 5) for the indicated times (mean ± SEM). j Intracellular CFU in macrophages from WT and Atp6v1e1 +/- mice treated with BMX inhibitor (10 nM) and infected with H37Rv (MOI = 5) for the indicated times (mean ± SEM). k–l Intracellular CFU and bacterial survival in macrophages from WT or Bmx +/- mice infected with H37Rv or H37RvΔChp2 for the indicated times (MOI = 5) (mean ± SEM). ( m )H&E staining of lung tissues from WT and Bmx +/- mice infected with H37Rv by intranasal infection ( ~ 200 CFU) for 4 weeks. (mean ± SEM, N = 3 mice) #1-3 indicate three representative lung tissues. Scale bar, 5000 μm. n Quantification of the histopathology score in (m) (mean ± SEM). o Bacterial CFUs in the lungs of WT and Bmx +/- mice in (m) (mean ± SEM). p Acid-fast staining of the lung tissues of WT and Bmx +/- mice in (m). Scale bar, 100 μm. Data in ( a–m ) are representative of one experiment with at least three independent biological replicates ( h–l , n , o , n = 3). Two-tailed unpaired Student’s t test (h-l and n) was used for statistical analysis. Two-sided Mann-Whitney U test ( o ) was used for statistical analysis.

Article Snippet: The siRNA targeting mouse Bmx (sc-38942) and control scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology, and a siRNA pool specifically targeting mouse Atp6v1e1 was obtained from RiboBio (Guangzhou, China).

Techniques: Western Blot, Phospho-proteomics, In Vitro, Purification, Transfection, Infection, Fractionation, Membrane, Staining, Fluorescence, Histopathology, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Mycobacterium tuberculosis modulates phosphorylation of host ATP6V1E1 to promote intracellular survival

doi: 10.1038/s41467-026-69331-1

Figure Lengend Snippet: a Detection ATP6V1E1 phosphorylation in macrophages infected with H37Rv or H37RvΔChp2 (MOI = 5) for indicated times. ratio: p-ATP6V1E1 Y56/57 /ATP6V1E1. b Immunoblotting of the lysates of macrophages transfected with scrambled or Bmx -specific siRNA and infected with H37Rv or H37RvΔChp2 (MOI = 5) for the indicated times by p-ATP6V1E1 Y56/Y57 antibody. ratio: p-ATP6V1E1 Y56/57 /ATP6V1E1. c Immunoblot analysis of ATP6V1E1 phosphorylation in peritoneal macrophages isolated from wild-type or Bmx +/- mice infected with H37Rv or H37RvΔChp2 for the indicated times (MOI = 5). ratio: p-ATP6V1E1 Y56/57 /ATP6V1E1. d In vitro phosphorylation assay of purified GFP-BMX and Flag-ATP6V1E1, which were overexpressed in HEK293T cells and treated with recombinant Chp2 protein(1 μg). e Peritoneal macrophages were infected with H37Rv or H37RvΔChp2 (MOI = 5) for 6 h, and changes of V-ATPase subunit abundance in the cytosolic (C) and membrane (M) fractions were analyzed by western blot. f Quantification of Western blots described in ( e ) (mean ± SEM). g , h Intracellular CFU and bacterial survival in macrophages transfected with scrambled or Bmx -specific siRNA and infected with H37Rv or H37RvΔRv1184c for the indicated times (MOI = 5) or then treat with NH 4 Cl (20 μM) (mean ± SEM), g P = 4.8736E-06 (Ctrl, si-Ctrl, 24 h). h P = 4.86583E-05 (Ctrl, si-Ctrl). ( i ) Surface plasmon resonance (SPR) assay of the direct interaction of ATP6V1E1 with BMX. BMX captured on a carboxy (COOH) chip can bind ATP6V1E1 with an affinity constant of 190 nM as determined in a localized surface plasmonic resonance (LSPR) assay. j Immunoblotting and immunoprecipitation of lysates from HEK293T cells transfected with plasmids for 48 h were performed and detected using the p-ATP6V1E1 Y56/Y57 antibody. k Endogenous interaction of ATP6V1E1 with BMX in mice peritoneal macrophages infected with H37Rv or H37RvΔRv1184c (MOI = 5) for the indicated times. l The predicted structural model of Rv1184c-BMX-V-ATPase complex by AlphaFold3 Server. Yellow represents ATP6V1G1, gray represents ATP6V1E1, blue represents Rv1184c, light purple represents BMX, dark purple represents the catalytic region of BMX, and red box represents the Y56/57 site of ATP6V1E1. Data in ( a–k ) are representative of one experiment with at least three independent biological replicates ( f–h , n = 3). Two-tailed unpaired Student’s t test ( f–h ) was used for statistical analysis.

Article Snippet: The siRNA targeting mouse Bmx (sc-38942) and control scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology, and a siRNA pool specifically targeting mouse Atp6v1e1 was obtained from RiboBio (Guangzhou, China).

Techniques: Phospho-proteomics, Infection, Western Blot, Transfection, Isolation, In Vitro, Purification, Recombinant, Membrane, SPR Assay, Immunoprecipitation, Two Tailed Test

Journal: bioRxiv

Article Title: Encephalitic Alphavirus Infection Induces PARP-1 Hyperactivation Mediated Energy Collapse in Motor Neurons

doi: 10.64898/2026.01.23.701280

Figure Lengend Snippet: ( A ) Representative western blot analysis of PAR accumulation and PARP-1 expression at 24 hpi with VEEV (MOI 1.0). NSC34 cells were transfected with either PARP1 siRNA (siPARP1; 20 pmol) or scrambled control siRNA (siCon; 20 pmol) 48 hours prior to infection. Pharmacological inhibition was achieved using ABT-888 (PARPi; 20µM) initiated 1 hour prior to infection and maintained throughout the experiment. ( B ) VEEV replication kinetics in NSC34 and diMN cells (MOI 1.0) following PARP-1 manipulation or nicotinamide riboside (NR; 500 µM) supplementation. To maintain effective concentrations, a 250µM NR spike was added to the infectious media at 12 hpi. Viral titers were quantified by plaque assay on Vero cells from supernatants collected between 6 and 48 hpi. Data represent mean ± SEM from at least three independent biological replicates. PARP-1 inhibition (siPARP1 and PARPi) resulted in an approximate one-log increase in viral titers between 12 and 24 hpi. (C–E) Metabolic and viability rescue at 24 hpi. (C) Intracellular NAD + (NAD/NADH-Glo) and (D)ATP levels (Luminescent ATP Detection) were quantified following treatment with siCon, siPARP1, PARPi, or NR as described above. All metabolic values were normalized to the number of viable cells in matched wells on the same plate to account for infection-induced cell loss. (E) Cell viability was assessed via Alamar Blue fluorescence. All values are expressed relative to mean of uninfected Mock controls. Statistical significance was determined by ordinary one-way ANOVA assuming a lognormal distribution; infected-treated groups were compared to the infected-untreated (or siCon) control via Dunnett’s multiple comparisons test. Mock and treatment-only controls are shown for reference. Data represent mean ± SEM., * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001

Article Snippet: A non-targeting scrambled siRNA (Santa Cruz, #sc-37007) served as a control.

Techniques: Western Blot, Expressing, Transfection, Control, Infection, Inhibition, Plaque Assay, Fluorescence