Journal: bioRxiv

Article Title: SUMO E1 covalent allosteric inhibitors modulate polyamine synthesis via the MAT2A-AdoMetDC axis

doi: 10.1101/2024.12.12.627095

Figure Lengend Snippet: Screening of new covalent inhibitors targeting the allosteric pocket of SUMO E1. (a) A schematic diagram of the SUMOylation pathway. SUMO E1 is a heterodimer of Aos1 and Uba2. Ubc9 is the sole SUMO E2 discovered thus far. (b) The complex of SUMO E1 and a covalent allosteric inhibitor (CAI) COH000 (PDB ID: 6CWY). COH000 is shown in sticks and formed a covalent bond with Uba2-C30. The residues of the allosteric pocket are shown in surface, and the residues of the ATP-binding pocket are shown in dotted surface. The SUMO-binding site is far away from the allosteric pocket and not shown. (c) MCULE-3064932370 and MCULE-6830015021 inhibited the formation of the SUMOylation of Uba2. Before the addition of SUMO1, the compounds were pre-incubated with SUMO E1 (Aos1/Uba2) at 37 °C for 30 minutes at various concentrations according to their solubility. MCULE-3064932370 and MCULE-6830015021 were 100 µM, MCULE-6321090433 was 50 µM, and the others were 500 µM. The reductant DTT (dithiothreitol) inhibits SUMOylation and was used as a control. The results with 60 min and 90 min pre-incubation had similar results (Figure S1, S2). All samples had 1% (v/v) DMSO. (d) Time-dependent and concentration-dependent inhibition of MCULE-3064932370 and MCULE-6830015021 on the SUMOylation of Uba2. The quantitative data from three replicates are shown in (e). Error bars represent mean ± s.d.. (f) Representative spectra from the full-protein mass spectrometry analysis. (g) Statistical analysis of the m/z data from multiple MALDI-TOF detections (n ≥ 6).

Article Snippet: The rabbit monoclonal anti-SUMO1 (Cat. NO. HY-P80351) was from MedChemExpress LLC (U.S.A.).

Techniques: Binding Assay, Incubation, Solubility, Control, Concentration Assay, Inhibition, Mass Spectrometry

Journal: bioRxiv

Article Title: SUMO E1 covalent allosteric inhibitors modulate polyamine synthesis via the MAT2A-AdoMetDC axis

doi: 10.1101/2024.12.12.627095

Figure Lengend Snippet: The binding of the new SUMO E1 CAIs depends on the Aos1/Uba2 complex. (a) Representative spectra and statistical analysis data from multiple MALDI-TOF detections (n ≥ 6) of the full-protein mass spectrometry analysis of the Uba2 protein when it was incubated with the inhibitors alone. (b) Representative spectra and statistical analysis data from multiple MALDI-TOF detections (n ≥ 6) of the full-protein mass spectrometry analysis of the Aos1 protein when it was incubated with the inhibitors alone. (c) The thermal denaturation result from the gel-based thermal shift assay of the Aos1/Uba2 complex with the new CAIs. (d) The competition experiment of the CAIs with the substrate SUMO1. (e) The 2D interaction diagrams of Aos1/Uba2 with COH000 (PDB ID: 6CWY), MCULE-3064932370, and MCULE-6830015021. Aos1 is the chain C, and Uba2 is the chain D. Since SCARdock only considers non-covalent interaction, the Uba2-C30 was computationally mutated to G30 as shown in the diagrams. Therefore, the ligands are shown not covalently bonded to Uba2. The ligands and residues are shown in ball-and-stick representation. The green dotted line indicates a hydrogen bond. The spoked arcs represent residues making nonbonded contacts with the ligands. The red circles indicate residues in equivalent 3D positions when the structural models are superposed.

Article Snippet: The rabbit monoclonal anti-SUMO1 (Cat. NO. HY-P80351) was from MedChemExpress LLC (U.S.A.).

Techniques: Binding Assay, Mass Spectrometry, Incubation, Thermal Shift Assay

Journal: bioRxiv

Article Title: SUMO E1 covalent allosteric inhibitors modulate polyamine synthesis via the MAT2A-AdoMetDC axis

doi: 10.1101/2024.12.12.627095

Figure Lengend Snippet: The new SUMO E1 CAIs perturb the SUMOylation pathway. T47D cells were treated with MCULE-3064932370 and MCULE-6830015021 for 48 h before evaluation. (a) The cellular levels of RanGAP1, RanGAP1-SUMO, and SUMO1 were assessed by Western blotting. (b) The cellular levels of Uba2 and Ubc9 were assessed by Western blotting.

Article Snippet: The rabbit monoclonal anti-SUMO1 (Cat. NO. HY-P80351) was from MedChemExpress LLC (U.S.A.).

Techniques: Western Blot

Journal: Cell Biology and Toxicology

Article Title: SENP1 inhibits aerobic glycolysis in Aβ 1-42 -incubated astrocytes by promoting PUM2 deSUMOylation

doi: 10.1007/s10565-025-09986-6

Figure Lengend Snippet: SENP1 deSUMOylates PUM2 . ( a and b ) SENP1 deSUMOylates PUM2 in cells. HEK-293 T cells were transfected with V5-PUM2, His-SUMO1 ( a ), or His-SUMO2/3 ( b ) with or without Flag-SENP1 for 48 h and treated with MG132 for 6 h. The cells were subjected to pulldown (PD) using Ni 2+ -NTA bead under denaturing conditions, followed by western blot. ( c and d ) SENP1 deSUMOylates endogenous PUM2 in cells. HEK-293 T cells were transfected with His-SUMO1 ( c ) or His-SUMO2/3 ( d ) with or without Flag-SENP1 for 48 h and treated with MG132 for 6 h. The cells were subjected to pulldown (PD) using a Ni 2+ -NTA bead under denaturing conditions, followed by western blot. ( e ) SENP1 de-SUMOylated endogenous PUM2 in HEK293T cells, confirmed by the CO-IP method. HEK293T cells were transfected with indicated plasmids (shNC, shSENP1), then lysed for the CO-IP method with an anti-SUMO1 antibody, an anti-PUM2 antibody, or normal IgG, followed by western blot assays with anti-PUM2 and anti-SUMO1 antibodies

Article Snippet: Centrifuge the cell lysate with 15,000 g for 5 min. Incubate the supernatant with the anti-PUM2 (ab92390, Abcam, MA, USA) (or anti-SUMO1(ab32058, Abcam, MA, USA)) conjugated A/G magnetic beads (B23201, Bimake, Shanghai, CN) overnight at 4 °C.

Techniques: Transfection, Western Blot, Co-Immunoprecipitation Assay

Journal: Cell Biology and Toxicology

Article Title: SENP1 inhibits aerobic glycolysis in Aβ 1-42 -incubated astrocytes by promoting PUM2 deSUMOylation

doi: 10.1007/s10565-025-09986-6

Figure Lengend Snippet: Glycolysis was inhibited by SENP1 through the reduction of PUM2 deSUMOylation in the Aβ 1–42 -incubated astrocytes . ( a ) Effects of Aβ 1–42 on SENP1 protein level by western blot. ( b ) Immunofluorescence staining analysis of the location of PUM2 (green) in both the nucleus and the cytoplasm of Aβ 1–42 -incubated astrocytes. The scale bar represents 25 μm. ( c ) Effects of PUM2 on ECAR in Aβ 1–42 -incubated astrocytes. ECAR is shown in milli pH per minute (milli pH/min), representing the rate of acidification of the extracellular medium. (d-e) Effects of PUM2 on lactate production ( d ) and glucose uptake ( e ) in Aβ 1–42 -incubated astrocytes. Data are presented as mean ± SD (n = 3, each). ** P < 0.01 versus the shNC group. ## P < 0.01 versus the shPUM2 + PUM2-NC group. ( f ) Effects of PUM2 on protein levels of HK1 and GLUT1 in Aβ 1–42 -incubated astrocytes by western blot. ( g ) Effects of PUM2 knockdown or re-expression in astrocytes on apoptosis of co-cultured neurons in the Aβ 1–42 -microenvironment. ( h ) Immunofluorescence staining analysis of the subcellular location of SUMO1 (red) and SENP1 (green) in Aβ 1–42 -incubated astrocytes. The scale bar represents 25 μm. ( i ) SENP1 deSUMOylates PUM2 in Aβ 1–42 -incubated astrocytes, confirmed by the CO-IP method. Aβ 1–42 -incubated astrocytes were transfected with indicated plasmids (shNC, shSENP1), then lysed for the CO-IP method with an anti-SUMO1 antibody, an anti-PUM2 antibody or normal IgG, followed by western blot assays with anti-PUM2 and anti-SUMO1 antibodies

Article Snippet: Centrifuge the cell lysate with 15,000 g for 5 min. Incubate the supernatant with the anti-PUM2 (ab92390, Abcam, MA, USA) (or anti-SUMO1(ab32058, Abcam, MA, USA)) conjugated A/G magnetic beads (B23201, Bimake, Shanghai, CN) overnight at 4 °C.

Techniques: Incubation, Western Blot, Immunofluorescence, Staining, Knockdown, Expressing, Cell Culture, Co-Immunoprecipitation Assay, Transfection

Journal: Science Advances

Article Title: Morphogen-induced kinase condensates transduce Hh signal by allosterically activating Gli

doi: 10.1126/sciadv.adq1790

Figure Lengend Snippet: ( A ) Top, immunostaining of HA-Fu in Fu-depleted Cl8 cells stably expressing RNAi-insensitive HA-Fu and treated with Hh-conditioned medium for the indicated time. Bottom, quantification of HA-Fu condensates in (A) from n = 10 cells for each time point. Data are means ± SD. * P < 0.05, ** P < 0.01 ( t test). ( B ) Immunostaining of Ci and HA-Fu proteins in Fu-depleted Cl8 cells stably expressing the indicated RNAi-insensitive HA-Fu constructs and treated with Hh-conditioned or control medium for 16 hours. ( C ) Left, schematic drawing of the sedimentation assay. Right, WB analyses of Fu, Ci, Sufu, phosphorylated Fu, and Ci in S3, S100, and P100 fractions from Fu-depleted Cl8 cells stably expressing the indicated RNAi-insensitive HA-Fu constructs and treated with Hh-conditioned or control medium. ( D ) Left, schematic drawing of in vitro condensation of Fu driven by phosphorylation and SUMOylation. Right, Flag-tagged kinase dead Fu (Fg-Fu K33R ) purified from Sf9 cells was subjected to in vitro kinase assay by FuN EE and CK1, followed by in vitro SUMOylation assay in the presence or absence of SUMO1 for the indicated periods of time. The distribution of Fg-Fu K33R in S100 and P100 at the indicated time points was analyzed by immunoblot after centrifugation. ( E ) Left, schematic drawing of in vitro deSUMOylation and de-condensation of Fu. Right, Flag-Fu EE condensates (p100) isolated from Sf9 cells were resuspended and deSUMOylated with WT or enzymatic dead (C580S) Ulp1C204 fused to GST for the indicated periods of time. The distribution of total Fu and phosphorylated Fu in condensates (P100) and supernatant (S100) at different time points was analyzed by immunoblot after re-centrifugation.

Article Snippet: Antibodies used in this study were as follows: mouse anti-Flag (M2, F3165, MilliporeSigma), mouse anti-HA (F7, Santa Cruz Biotechnology), rabbit anti-HA (ab9110, Abcam), mouse anti-Myc (9E10, Santa Cruz Biotechnology), rabbit anti-Myc (ab9106, Abcam), rat anti-Ci (2A1, DSHB), mouse anti-Fu (22F10, DSHB), mouse anti-Sufu (25H3, DSHB), mouse anti-Patched (Apa1, DSHB), mouse anti-engrailed (4D9, DSHB), mouse anti–α-tubulin (T9026, MilliporeSigma), rabbit anti-histone3 (ab1791, Abcam), goat anti-Gli2 (AF3635, R&D systems), rabbit anti-SUMO1 (4930, Cell Signaling Technology), mouse anti-acetylated tubulin (T7451, MilliporeSigma), mouse anti-ubiquitin (P4D1, Santa Cruz Biotechnology), and rabbit anti-Sufu (ab28083, Abcam).

Techniques: Immunostaining, Stable Transfection, Expressing, Construct, Control, Sedimentation, In Vitro, Purification, Kinase Assay, Western Blot, Centrifugation, Isolation

Journal: Science Advances

Article Title: Morphogen-induced kinase condensates transduce Hh signal by allosterically activating Gli

doi: 10.1126/sciadv.adq1790

Figure Lengend Snippet: ( A ) WB analysis of endogenous Fu maturation and Ci phosphorylation in S100 and P100 fractions from Cl8 cells treated with Hh-conditioned medium for the indicated periods of time. ( B ) In vitro maturation assay of Fu in the absence or presence of NEM (deSUMOylation inhibitor) or TAK981(SUMOylation inhibitor). ( C ) In vitro maturation assay of Fu. Flag-Fu EE proteins expressed in Sf9 cells at high MOI were separated into S100 and P100 fractions. Purified (S100) or resuspended (P100) Flag-Fu EE proteins were incubated with MPA (Sf9 cell lysate, ATP, and SUMO1) for the indicated periods of time. The distribution of Fu in condensates (P100) and supernatant (S100) at different time points after incubation was analyzed by immunoblot after re-centrifugation. ( D ) In vitro Fu maturation and Ci phosphorylation. Flag-Fu EE proteins expressed in Sf9 cells at high MOI were separated into S100 and P100 fractions and then incubated with or without MPA in the presence of Sufu-Ci complexes purified from Sf9 cells for 6 hours, followed centrifugation into S100 and P100 fractions and immunoblot analysis with the indicated antibodies. ( E ) Left, Flag-tagged Fu EE or Fu∆Sufu EE condensates (P100) derived from Sf9 cells were resuspended and incubated with MPA in the presence of free Ci or Sufu-Ci purified from Sf9 cells, followed by immunoblot analysis with the indicated antibodies. Right, Fu∆Sufu can undergo maturation but fail to phosphorylate Sufu-bound Ci although it can phosphorylate free Ci. ( F ) Left, Flag-Fu EE was purified from S100 after in vitro maturation experiments and then incubated with either free Ci or Sufu-Ci, followed by immunoblot analysis with the indicated antibodies. Right, the released mature Fu is unable to phosphorylate Sufu-bound Ci but can still phosphorylate free Ci. ( G ) Model of Ci phosphorylation driven by Fu maturation in Fu condensates.

Article Snippet: Antibodies used in this study were as follows: mouse anti-Flag (M2, F3165, MilliporeSigma), mouse anti-HA (F7, Santa Cruz Biotechnology), rabbit anti-HA (ab9110, Abcam), mouse anti-Myc (9E10, Santa Cruz Biotechnology), rabbit anti-Myc (ab9106, Abcam), rat anti-Ci (2A1, DSHB), mouse anti-Fu (22F10, DSHB), mouse anti-Sufu (25H3, DSHB), mouse anti-Patched (Apa1, DSHB), mouse anti-engrailed (4D9, DSHB), mouse anti–α-tubulin (T9026, MilliporeSigma), rabbit anti-histone3 (ab1791, Abcam), goat anti-Gli2 (AF3635, R&D systems), rabbit anti-SUMO1 (4930, Cell Signaling Technology), mouse anti-acetylated tubulin (T7451, MilliporeSigma), mouse anti-ubiquitin (P4D1, Santa Cruz Biotechnology), and rabbit anti-Sufu (ab28083, Abcam).

Techniques: In Vitro, Purification, Incubation, Western Blot, Centrifugation, Derivative Assay

Journal: Science Advances

Article Title: Morphogen-induced kinase condensates transduce Hh signal by allosterically activating Gli

doi: 10.1126/sciadv.adq1790

Figure Lengend Snippet: ( A and B ) Representative images of immunostaining (top) and quantification (bottom; n = 15 cells) of ciliary-localized Ulk3-HA and Flag-Gli2 (A) or Sufu (B) in NIH3T3 cells expressing endogenously tagged Ulk3 and Gli2 and treated with Shh for the indicated time. Data are means ± SD. * P < 0.05, ** P < 0.01 ( t test). Acetylated tubulin (AcTub) staining marks the primary cilia. ( C ) Representative images of immunostaining of ciliary-localized Ulk3-HA and SUMO1 in NIH3T3 Ulk3-HA+Flag-Gli2 cells treated with or without Shh. ( D ) Representative images of immunostaining of ciliary-localized Ulk3-HA and SUMO1 in NIH3T3 DKO cells infected with or without lentiviral Ulk3-HA and treated with Shh. ( E ) Representative images of immunostaining (left) and quantification (right, n = 15 cells) of ciliary-localized Ulk3 and Gli2 in Ulk3/Stk36 DKO and Gli2 depleted NIH3T3 cells expressing Flag-Gli2 and the indicated Ulk3-HA constructs and treated with or without Shh. Data are means ± SD. *** P < 0.001 ( t test). ( F ) Immunostaining to examine the ciliary Gli2 phosphorylation (pGli2) in NIH3T3 cells transfected with Myc-Gli2 WT or Myc-Gli2 SA and Flag-Sufu and treated with or without Shh. ( G ) NIH3T3 cells expressing Ulk3-HA and Flag-Gli2 at endogenous loci were treated with control (eGFP) or Kapβ2 shRNA in the presence or absence of Shh, followed by immunostaining to examine Ulk3/Gli2 ciliary localization (top) or IP and WB analysis to examine Ulk3 maturation and Gli2 phosphorylation (bottom). ( H ) Schematic drawing of Ulk3 condensation at ciliary tip that drives Ulk3 maturation and Gli2 activation.

Article Snippet: Antibodies used in this study were as follows: mouse anti-Flag (M2, F3165, MilliporeSigma), mouse anti-HA (F7, Santa Cruz Biotechnology), rabbit anti-HA (ab9110, Abcam), mouse anti-Myc (9E10, Santa Cruz Biotechnology), rabbit anti-Myc (ab9106, Abcam), rat anti-Ci (2A1, DSHB), mouse anti-Fu (22F10, DSHB), mouse anti-Sufu (25H3, DSHB), mouse anti-Patched (Apa1, DSHB), mouse anti-engrailed (4D9, DSHB), mouse anti–α-tubulin (T9026, MilliporeSigma), rabbit anti-histone3 (ab1791, Abcam), goat anti-Gli2 (AF3635, R&D systems), rabbit anti-SUMO1 (4930, Cell Signaling Technology), mouse anti-acetylated tubulin (T7451, MilliporeSigma), mouse anti-ubiquitin (P4D1, Santa Cruz Biotechnology), and rabbit anti-Sufu (ab28083, Abcam).

Techniques: Immunostaining, Expressing, Staining, Infection, Construct, Transfection, Control, shRNA, Activation Assay

Journal: PLOS Biology

Article Title: SUMO-mediated regulation of H3K4me3 reader SET-26 controls germline development in C . elegans

doi: 10.1371/journal.pbio.3002980

Figure Lengend Snippet: Antibodies and dilutions used in this study.

Article Snippet: anti-SUMO-1 21C7 (1:1,000) , DSHB Cat# SUMO-1 21C7, RRID:AB_2198257.

Techniques:

Journal: Nature Communications

Article Title: Viral RNA polymerase as a SUMOylation decoy inhibits RNA quality control to promote potyvirus infection

doi: 10.1038/s41467-024-55288-6

Figure Lengend Snippet: a qRT-PCR analysis of the Pelota expression level in Arabidopsis rosette and cauline leaves under TuMV infection. The error bars indicate mean ± SD ( n = 3 biologically independent samples with averaged technical duplicates shown), and * p = 0.0318, ns means not significant ( p = 0.0701) ( p values determined by two-tailed Student’s t test ). b In vivo SUMOylation analysis of Pelota. Proteins were immunoprecipitated with anti-GFP beads from extracts of Pelota: pelota transgenic plants. SUMOylated Pelota was detected with anti-SUMO1 antibody. TuMV accumulation was detected with anti-CP antibodies. CBB-staining of RbcL was a loading control. Independent experiments were performed three times with similar results. The samples were derived from the same experiment and that blots were processed in parallel. c In vivo SUMOylation analysis of Pelota. Pelota-YFP and Myc-SUMO1 co-expressed with a gradient amount of TuMV in N. benthamiana leaves. Samples were treated with MG132 for 6 h before collected at 60 h post-infiltration (hpi). Extracted proteins were immunoprecipitated with anti-GFP beads, and SUMOylated Pelota and total proteins were detected with anti-Myc antibodies. TuMV accumulation was detected with anti-CP antibodies. CBB-staining of RbcL was a loading control. Quantification of protein levels was performed using ImageJ software. Independent experiments were performed three times with similar results. The samples were derived from the same experiment and that gels/blots were processed in parallel. d qRT-PCR analysis of P3 RNA accumulation during expression of Myc-GUS (GUS) or Myc-Pelota (Pelota) in TuMV infected N. benthamiana leaves at 40 hpi. The error bars indicate mean ± SD ( n = 3 biologically independent samples with averaged technical duplicates shown), and ** p = 0.0005, ns means not significant ( p = 0.7360) ( p values determined by two-tailed Student’s t test). e Competitive Co-IP assays in vivo. Pelota-YFP and Hbs1-Flag were co-expressed with a gradient amount of TuMV in N. benthamiana leaves. Note: samples were treated with MG132 for 6 h before collection to exclude the variations from protein degradation, and immunoprecipitated with anti-GFP beads. The immunoprecipitated and input proteins were then analyzed with anti-Flag and anti-GFP antibodies. CBB-staining of RbcL was a loading control. Quantification of protein levels was performed using ImageJ software. Independent experiments were performed three times with similar results. The samples were derived from the same experiment and that blots were processed in parallel.

Article Snippet: Equal amount of eluted proteins were analyzed by immunoblotting with anti-Flag from Abmart (1:5000, M20026), anti-MBP from TransGen (1:5000, HT701), anti-His from TransGen (1:5000, HT501), anti-GST from TransGen (1:5000, HT601) and anti-SUMO1 (ABclonal Technology), Goat Anti-Rabbit/Mouse IgG (H + L)-HRP Conjugated (EASYBIO, BE0102/BE0102, 1:10000) antibodies.

Techniques: Quantitative RT-PCR, Expressing, Infection, Two Tailed Test, In Vivo, Immunoprecipitation, Transgenic Assay, Staining, Control, Derivative Assay, Software, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Viral RNA polymerase as a SUMOylation decoy inhibits RNA quality control to promote potyvirus infection

doi: 10.1038/s41467-024-55288-6

Figure Lengend Snippet: a In vivo SUMOylation of Pelota. Pelota-YFP and Myc-SUMO1 were co-expressed with Flag-tagged TuMV-encoded proteins in N. benthamiana leaves, and Pelota was immunoprecipitated with anti-GFP beads at 60 hpi. Immunoprecipitated proteins were then analyzed with anti-Myc, anti-Flag and anti-GFP antibodies. Stars indicate the Flag-tagged GUS and viral encoded proteins (P1, P3, CI, VPg, NIa, NIb, and CP). This experiment was repeated three times showing similar results. The samples were derived from the same experiment and that blots were processed in parallel. b Pelota-YFP and Myc-SUMO1 were co-expressed with a gradient amount of NIb in N. benthamiana leaves. Note: samples were treated with MG132 for 6 h before collection to exclude the variations from protein degradation, and proteins were immunoprecipitated with anti-GFP beads at 60 hpi. Immunoprecipitated and input proteins were then analyzed with anti-Myc, anti-Flag, and anti-GFP antibodies. CBB-staining of RbcL was a loading control. Quantification of protein levels was performed using ImageJ software. Independent experiments were performed three times with similar results. The samples were derived from the same experiment and that blots were processed in parallel. c Growth phenotype of Col-0 and P3-OE #4 transgenic Arabidopsis plants. d qRT-PCR analysis of P3 RNA accumulation in P3-OE #4 transgenic Arabidopsis protoplasts. GFP, GFP-Pelota or GFP-Pelota with GFP-NIb were co-transformed in P3-OE #4 Arabidopsis protoplasts. The error bars indicate mean ± SD ( n = 3 biologically independent samples with averaged technical duplicates shown). p values determined by one-way ANOVA and Dunnett testing, ** p = 0.0091, ns means not significant ( p = 0.1030). e qRT-PCR analysis of P3 RNA accumulation at 40 hpi during expression of GUS and Pelota in N. benthamiana leaves transfected with buffer and NIb. The error bars indicate mean ± SD ( n = 3 biologically independent samples with averaged technical duplicates shown), and ** p = 0.0001, ns means not significant ( p = 0.8688) ( p values determined by two-tailed Student’s t test ). f Virus symptoms and GFP fluorescence in TuMV-GFP infected plants after inoculation with GUS (mock), Pelota, Pelota+NIb. Plants were photographed under ambient or UV light at 8 dpi. g Analysis of TuMV-GFP RNA accumulations indicated in ( f ), systemically infected leaves. The error bars indicate mean ± SD ( n = 3 biologically independent samples with averaged technical duplicates shown). p values determined by one-way ANOVA and Dunnett testing, ** p = 0.0020, ns means not significant ( p = 0.9743).

Article Snippet: Equal amount of eluted proteins were analyzed by immunoblotting with anti-Flag from Abmart (1:5000, M20026), anti-MBP from TransGen (1:5000, HT701), anti-His from TransGen (1:5000, HT501), anti-GST from TransGen (1:5000, HT601) and anti-SUMO1 (ABclonal Technology), Goat Anti-Rabbit/Mouse IgG (H + L)-HRP Conjugated (EASYBIO, BE0102/BE0102, 1:10000) antibodies.

Techniques: In Vivo, Immunoprecipitation, Derivative Assay, Staining, Control, Software, Transgenic Assay, Quantitative RT-PCR, Transformation Assay, Expressing, Transfection, Two Tailed Test, Virus, Fluorescence, Infection

Journal: Nature Communications

Article Title: Viral RNA polymerase as a SUMOylation decoy inhibits RNA quality control to promote potyvirus infection

doi: 10.1038/s41467-024-55288-6

Figure Lengend Snippet: a Predicted SUMO interacting motifs (SIMs) on the NIb protein. Replacement of the Valine (V), Isoleucine (I), and Leucine (L) with the Alanine (A) amino acid resulted in SIM mutations. b Identification of the interaction between SCE1 and the NIb (sim mutants) in yeast. Yeast cells co-expressing BD-SCE1 and AD-EV were used as negative control. c MBP-SCE1 protein combined with GST-Pelota was incubated with Flag-NIb (or sim mutants) in vitro. The proteins were immunoprecipitated with Amylose Resin. Input and pull-down proteins were analyzed with anti-GST, Flag, and MBP antibodies. This experiment was repeated three times showing similar results. The samples were derived from the same experiment and that blots were processed in parallel. d BiFC assay showing interactions between Pelota, NIb, NIb sim2 and NIb sim3 in RFP-H2B (a nuclear marker, the red fluorescent protein fused to histone 2B) transgenic N. benthamiana leaves. Scale bars correspond to 20 μm. This experiment was repeated three times showing similar results. e Competitive Co-IP assays in vivo. SCE1-YFP and Myc-Pelota were co-expressed with NIb (or sim mutants)-Flag in N. benthamiana leaves. Note: samples were treated with MG132 for 6 h before collection to exclude the variations from protein degradation, and proteins were immunoprecipitated with anti-GFP beads. The immunoprecipitated and input proteins were then analyzed with anti-Myc, anti-Flag, and anti-GFP antibodies. CBB-staining of RbcL was a loading control. Quantification of protein levels was performed using ImageJ software. Independent experiments were performed three times with similar results. The samples were derived from the same experiment and that blots were processed in parallel. f In vivo SUMOylation assay. Pelota-YFP and Myc-SUMO1 were co-expressed with NIb (or sim mutants)-Flag in N. benthamiana leaves. The proteins were immunoprecipitated with anti-GFP beads at 40 hpi and analyzed with anti-Myc, anti-Flag and anti-GFP antibodies. This experiment was repeated three times showing similar results. The samples were derived from the same experiment and that blots were processed in parallel. g Phenotypes of N. benthamiana plants infected by TuMV, TuMV-NIb sim1 , TuMV-NIb sim2 and TuMV-NIb sim3 at 10 dpi. Bar = 5 cm. h Phenotypes of B. napus plants infected by TuMV, TuMV-NIb sim1 , TuMV-NIb sim2 and TuMV-NIb sim3 at 15 dpi. Bar = 5 cm. i qRT-PCR analysis of TuMV, TuMV-NIb sim1 , TuMV-NIb sim2 and TuMV-NIb sim3 genomic RNA accumulation in ( g , h ). TuMV-GFP-NIb sim2 genomic RNA accumulation was normalized to 1. The error bars indicate mean ± SD ( n = 3 biologically independent samples with averaged technical duplicates shown). p values determined by one-way ANOVA and Tukey testing, *** p < 0.0001 (TuMV or TuMV-NIb sim1 vs TuMV-NIb sim2 or TuMV-NIb sim3 ). ND means not detected.

Article Snippet: Equal amount of eluted proteins were analyzed by immunoblotting with anti-Flag from Abmart (1:5000, M20026), anti-MBP from TransGen (1:5000, HT701), anti-His from TransGen (1:5000, HT501), anti-GST from TransGen (1:5000, HT601) and anti-SUMO1 (ABclonal Technology), Goat Anti-Rabbit/Mouse IgG (H + L)-HRP Conjugated (EASYBIO, BE0102/BE0102, 1:10000) antibodies.

Techniques: Expressing, Negative Control, Incubation, In Vitro, Immunoprecipitation, Derivative Assay, Bimolecular Fluorescence Complementation Assay, Marker, Transgenic Assay, Co-Immunoprecipitation Assay, In Vivo, Staining, Control, Software, Infection, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Viral RNA polymerase as a SUMOylation decoy inhibits RNA quality control to promote potyvirus infection

doi: 10.1038/s41467-024-55288-6

Figure Lengend Snippet: a Comprehensive analysis of NIb sequences from 12 genera in the Potyviridae family, featuring a phylogenetic tree, multi-alignment of conserved sites, and a conservation heatmap. The tree branches depict the genetic divergence, with branch lengths indicating the degree of sequence variation. The multi-alignment of conserved sites showcases three SIM sites across the viruses. The right bubble illustrates the conservation levels of these sites across these virus sequences, with color intensity and bubble size representing the degree of conservation. b BiFC assay showing interactions between SCE1 and PVY, PVMV, and ANRSV NIb (as well as NIb sim mutants) in RFP-H2B (a nuclear marker, the red fluorescent protein fused to histone 2B) transgenic N. benthamiana leaves. Scale bars correspond to 20 μm. This experiment was repeated three times showing similar results. c Analysis of the interactions between SCE1 and PVY, PVMV, and ANRSV NIb (as well as NIb sim mutants) in yeast. d In vivo SUMOylation of Pelota. Pelota-YFP and Myc-SUMO1 were co-expressed with PVY, PVMV, and ANRSV NIb (as well as sim mutants) in N. benthamiana leaves, and Pelota was immunoprecipitated with anti-GFP beads at 40 hpi. Immunoprecipitated proteins were then analyzed with anti-Myc and anti-GFP antibodies. This experiment was repeated three times showing similar results. The samples were derived from the same experiment and that blots were processed in parallel. e qRT-PCR analysis of P3 RNA accumulation at 40 hpi during expression of GUS or Pelota with PVY, PVMV, and ANRSV NIb (as well as NIb sim mutants) in N. benthamiana leaves. The error bars indicate mean ± SD ( n = 3 biologically independent samples with averaged technical duplicates shown). p values determined by one-way ANOVA and Dunnett testing, *** p < 0.001.

Article Snippet: Equal amount of eluted proteins were analyzed by immunoblotting with anti-Flag from Abmart (1:5000, M20026), anti-MBP from TransGen (1:5000, HT701), anti-His from TransGen (1:5000, HT501), anti-GST from TransGen (1:5000, HT601) and anti-SUMO1 (ABclonal Technology), Goat Anti-Rabbit/Mouse IgG (H + L)-HRP Conjugated (EASYBIO, BE0102/BE0102, 1:10000) antibodies.

Techniques: Sequencing, Virus, Bimolecular Fluorescence Complementation Assay, Marker, Transgenic Assay, In Vivo, Immunoprecipitation, Derivative Assay, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

doi: 10.1038/s41467-024-55034-y

Figure Lengend Snippet: a Ni 2+ -NTA bead affinity pull-down assay results showing the modification of exogenous huANP32A (left) and huANP32B (right) with SUMO1, SUMO2, and SUMO3 in HEK293T cells. b Ni 2+ -NTA bead affinity pull-down assay results showing the modification of endogenous huANP32A and huANP32B with SUMO1 in HEK293T cells. *indicates non-specific protein band. c Western blot analysis showing that H9N2 virus infection upregulates the global SUMOylation level in HEK293T cells. d Ni 2+ -NTA bead affinity pull-down assay results showing that the SUMOylation of huANP32A (left) or huANP32B (right) in HEK293T cells was notably enhanced by H9N2 AIV infection. HEK293T cells were transfected with the indicated plasmids for 24 hours followed by H9N2 AIV infection (MOI = 0.01) for another 24 hours. Cells were then harvested for SUMOylation assays and immunoblotting analysis. In ( a to d ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

Techniques: Pull Down Assay, Modification, Western Blot, Virus, Infection, Transfection

Journal: Nature Communications

Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

doi: 10.1038/s41467-024-55034-y

Figure Lengend Snippet: a Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated constructs on H9N2 vPol activity. Values above K0 were statistically analyzed using one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32A-K0 (error bars represent the mean ± SD of n = 4 independent biological replicates). b Schematic representation of the huANP32A-K0 mutants generated. Western blots demonstrate comparable expression levels for all indicated constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of MDCK-TKO cells stably reconstituted with the indicated huANP32A-K0-Flag constructs or empty vector. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K153 sites of huANP32A can be modified by SUMO1. h Co-IP experiments showing that the K68R/K153R mutations in huANP32A suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32A. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-Flag or huANP32A-K68R/K153R-Flag (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32A-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). In ( g , h ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

Techniques: Construct, Activity Assay, Generated, Western Blot, Expressing, Virus, Control, Stable Transfection, Mutagenesis, Plasmid Preparation, Pull Down Assay, Modification, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

doi: 10.1038/s41467-024-55034-y

Figure Lengend Snippet: a Minigenome assays in HEK293T-TKO cells comparing the effect of indicated huANP32B-K0 constructs on the H9N2 vPol activity. Values higher than K0 were analyzed via one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32B-K0 (error bars represent the mean ± SD of n = 3 independent biological replicates). b Schematic representation of the huANP32B-K0 mutants generated. Western blots confirmed comparable expression levels of all constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32B-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32B-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32B-K0-Flag constructs. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K116 sites of huANP32B can be modified by SUMO1. h Co-IP experiments showing that the K68R/K116R mutations in huANP32B suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32B constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32B. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32B-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32B-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). Experiments in ( g and h ) were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

Techniques: Construct, Activity Assay, Generated, Western Blot, Expressing, Virus, Control, Stable Transfection, Mutagenesis, Pull Down Assay, Modification, Co-Immunoprecipitation Assay, Two Tailed Test