Journal: Nucleic Acids Research

Article Title: Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation

doi: 10.1093/nar/gkm617

Figure Lengend Snippet: Proteins analyzed for SUMOylation by UFDS

Article Snippet: Antibodies against the following proteins or peptides were used: Ubc9 (H81, Santa Cruz) and GFP (B-2, Santa Cruz).

Techniques: Activation Assay, Binding Assay

Journal: Nucleic Acids Research

Article Title: Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation

doi: 10.1093/nar/gkm617

Figure Lengend Snippet: Identification of new SUMOylation substrates using UFDS. The fusion of Ubc9 to the C-terminus ( A ) or the N-terminus ( B ) of a substrate protein induces the E3-ligase independent SUMOylation of the substrate protein. The expression plasmids for the Ubc9 (U) fusion proteins were expressed alone or together with EGFP-SUMO1 (E-S1) in HEK293 cells. After 24–48 h protein extracts of the transfectants were analyzed by immunoblotting using an Ubc9 antibody. Examples for strong mono-SUMOylated (HMGN2, TAF10, CDC37 and FOS), strongly di-SUMOylated (HSF2BP and CSNK2B and ZNRD1), weakly (CDK4) and non-SUMOylated (ELLE3) Ubc9 fusion proteins are shown ( C , c.f. ). The bands for the Ubc9 fusion proteins are marked with P, the EGFP-SUMO1 conjugated Ubc9-fusion proteins with E-P Bands for double EGFP-SUMO1 conjugated Ubc9-fusion proteins are marked with 2E-P, Ubc9-fusion proteins modified at alternative SUMOylation sites are marked by black arrows. Bands representing Ubc9-fusion proteins modified by endogenous SUMO (CIAO1, CRSP9 and FOS) are marked with black asterisks.

Article Snippet: Antibodies against the following proteins or peptides were used: Ubc9 (H81, Santa Cruz) and GFP (B-2, Santa Cruz).

Techniques: Expressing, Western Blot, Modification

Journal: Nucleic Acids Research

Article Title: Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation

doi: 10.1093/nar/gkm617

Figure Lengend Snippet: p53 SUMOylation by Ubc9, SUMO-deconjugating enzyme ( A ) and SUMO ligases ( B ). (A and B) p53 was expressed alone or together with EGFP-SUMO1 and the indicated proteins in HEK293 cells. After 24 h, protein extracts of the transfectants were analyzed by immunoblotting using a p53 antibody (WB:p53). p53 and the p53 conjugated with endogenous SUMO (S) or with one EGFP-SUMO1 (E-S1) are indicated by black arrow heads. An unspecific band in A and B is marked by white arrows.

Article Snippet: Antibodies against the following proteins or peptides were used: Ubc9 (H81, Santa Cruz) and GFP (B-2, Santa Cruz).

Techniques: Western Blot

Journal: Nucleic Acids Research

Article Title: Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation

doi: 10.1093/nar/gkm617

Figure Lengend Snippet: Constitutive SUMOylation of newly identified substrate proteins without Ubc9 fusion. The expression plasmids for the GST fusion proteins G-FOS ( A ), G-CRSP9 ( B ), G-CDC37 ( C ) were transfected alone or together with expression plasmids for the indicated proteins in HEK293 cells. After 24 h, protein cell lysates were analyzed by immunoblotting using a GST antibody (WB: GST). The fusion proteins conjugated with endogenous SUMO (S) or with EGFP-SUMO1 (E-S1) are indicated by black arrow heads (A–C). For GST pull downs (pd) 24 h after transfection, the GST fusion proteins from the extracts of transfectants were purified on glutathione sepharose and analyzed in a western blot with a SUMO1 antibody (WB:SUMO1). Afterwards, the membranes were stripped and the fusion proteins were detected by western blot with a GST antibody (WB:GST). Additionally, the whole cell lysates (WCL) of the pull downs were analyzed for EGFP-SUMO1 expression by immunoblotting using a SUMO1 antibody (WB:SUMO1). The EGFP-SUMO1 protein is indicated by a black arrow head, the EGFP-SUMO1 conjugated proteins (E-S1-protein) are indicated by a black line. An unspecific band in B is indicated by a white arrow.

Article Snippet: Antibodies against the following proteins or peptides were used: Ubc9 (H81, Santa Cruz) and GFP (B-2, Santa Cruz).

Techniques: Expressing, Transfection, Western Blot, Purification

Journal: Nucleic Acids Research

Article Title: Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation

doi: 10.1093/nar/gkm617

Figure Lengend Snippet: Induced SUMOylation of substrate proteins without Ubc9 fusion. The expression plasmids for the GST fusion proteins G-CSNK2B ( A ), G-TAF10 ( B ), G-HSF2BP ( C ), G-PSMC3 ( D ) and G-DRG1 ( E ) were transfected alone or together with expression plasmids for the indicated proteins in HEK293 cells. After 24 h, protein extracts of the transfectants were analyzed by an immunoblot using a GST antibody (WB:GST). The fusion proteins conjugated with endogenous SUMO (S) or with EGFP-SUMO1 (E-S1) are indicated by black arrow heads (A–E). For GST pull downs (pd) 24 h after transfection, the GST fusion proteins from the extracts of transfectants were purified on glutathione sepharose and analyzed by a western blot with a SUMO1 antibody (WB:SUMO1). Afterwards, the membranes were stripped and the fusion proteins were detected by western blotting using a GST antibody (WB:GST). Additionally, the whole cell lysates (WCL) of the pull downs were analyzed for EGFP-SUMO1 expression by immunoblotting using a SUMO1 antibody (WB:SUMO1). The EGFP-SUMO1 protein is indicated by a black arrow head, the EGFP-SUMO1 conjugated proteins (E-S1-protein) are indicated by a black line. An unspecific band in A is marked by a white arrow.

Article Snippet: Antibodies against the following proteins or peptides were used: Ubc9 (H81, Santa Cruz) and GFP (B-2, Santa Cruz).

Techniques: Expressing, Transfection, Western Blot, Purification

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: SARS-COV-2 N protein interacts with UBC9. ( A ) pCDNA3.1 (3XFlag Tag) with N protein-coding genes and pEGFP-N1 (GFP Tag) with UBC9-coding genes were co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. ( B ) pDsRED-mono-N1 (RFP Tag) with SARS-COV-2 N protein-coding genes and pEGFP-N1 (GFP Tag) with UBC9 or UBC9 C93A coding genes were expressed or co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cells were stained with DAPI before confocal microscopy imaging. Endogenous UBC9 in HEK293T cells was also detected by FITC-conjugated Goat Anti-Mouse IgG(H + L), which recognizes anti-UBC9 mouse antibodies. Scale bars: 10 μm. ( C ) pET28a (HIS 6 tag, Kan + ) with SARS-COV-2 N protein coding genes and pGEX-6P-1 (GST tag, Amp + ) with UBC9 or UBC9 C93A coding genes were co-expressed in BL21 (DE3) E. coli . cell lysates were subjected to Co-IP after IPTG (1 mM) induction for 6 h. ( D ) pCDNA3.1 (3XFlag Tag) with SARS-COV-2 N protein-coding genes was over-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Transfection, Co-Immunoprecipitation Assay, Staining, Confocal Microscopy, Imaging

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: Human beta coronavirus N protein interacts with UBC9. ( A ) pET28a-N (HIS 6 tag, Kan + ) containing five human beta coronavirus N protein-coding genes and pGEX-UBC9 (GST tag, Amp + ) harboring the UBC9 coding gene were co-expressed in BL21 (DE3) E. coli Cell lysates were prepared and subjected to Co-IP following IPTG induction (1 mM) for 6 h. ( B ) pEGFP-N1 (GFP Tag) with three human beta coronavirus N protein-coding genes and pCDNA3.1 (3XFlag Tag) with the UBC9 coding gene were co-expressed in HEK293T cells. After a 24- h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Co-Immunoprecipitation Assay, Transfection

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: SARS-COV-2 N protein enhanced the molecular interaction between UBC9 and MAVS. ( A ) pEGFP-N1 (GFP Tag) with SARS-COV-2 N protein or UBC9 coding genes and pCDNA3.1 (3XFlag Tag) with MAVS coding gene were co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. ( B ) Relative Ubc9 band intensity before or after IP: Flag in ( A ) were calculated by using ImageJ in triplet replicates. ns: no significant difference, *** p < 0.001. ( C ) Schematic diagram of wild-type SARS-CoV-2 N protein and truncated mutants. ( D ) pEGFP-N1 (GFP Tag) with UBC9 coding genes and pCDNA3.1 (3XFlag Tag) with SARS-COV-2 N protein or truncation coding gene were co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Transfection, Co-Immunoprecipitation Assay

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: The expression of UBC9 in HEK293T, Vero, Vero E6, Huh7, and HRT18 cells was detected by Western blotting.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Expressing, Western Blot

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: The interaction between the SARS-CoV-2 N protein and UBC9 inhibits MAVS ubiquitination by enhancing its SUMOylation. ( A , B ) pEGFP-N1 (GFP Tag) with SARS-COV-2 N coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA, or K63-His6 coding genes were co-expressed in HEK293T cells (endogenous UBC9 highly expressed) or Vero E6 cells (endogenous UBC9 underexpressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: The interaction of N protein and UBC9 plays a crucial role in regulating the SUMOylation and ubiquitination modifications of MAVS. ( A ) pEGFP-N1 (GFP Tag) with SARS-COV-2 N coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA or SUMO3 KR -HA, K63-His 6 coding genes were co-expressed in HEK293T cells (endogenous UBC9 was highly expressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. ( B ) pEGFP-N1 (GFP Tag) with SARS-COV-2 N coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA, UBC9 or UBC9 C93A , K63-His 6 coding genes were co-expressed in Vero E6 cells (endogenous UBC9 underexpressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. ( C ) pEGFP-N1 (GFP Tag) with SARS-COV-2 N, N 44–419 or N 174–419 coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA, and K63-His 6 coding genes were co-expressed in HEK293T cells (endogenous UBC9 highly expressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: UBC9 plays a critical role in the process of impaired IFN I response caused by the in-teraction between the N protein and MAVS during virus infection. ( A , B ) pCDNA3.1 with SARS-CoV-2 N protein-coding genes was gradually increased in HEK293T cells or VERO E6 cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Western blotting. ( C , D ) The relative band intensity of Western blotting results (a relative band quantification of phosphorylation protein to total protein) in ( A , B ) were calculated by using ImageJ in triplet replicates. ns: no significant difference, p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Virus, Infection, Transfection, Western Blot, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms

doi: 10.3389/fimmu.2018.01142

Figure Lengend Snippet: Confirmation of differentially expressed proteins and phosphoproteins by western blotting and Phos-tag western Blotting. (A) Analysis of ubiquitin conjugating enzyme E2 I, ubiquitin conjugating enzyme E2 L3, glyceraldehyde-3-phosphate dehydrogenase, β-actin expression levels in foot-and-mouth disease virus (FMDV)-infected and control cells by western blotting. SILAC-ratios and immunoblotting ratios (infection/control) were shown on the right side. (B) Analysis of the dynamic phosphorylation alterations of the three differentially phosphoproteins (ribosomal protein L15, chromosome 5 open reading frame 24, and FOS-like 2) in FMDV-infected and control cells by Phos-tag western blotting.

Article Snippet: To confirm the expression levels of GAPDH, ubiquitin conjugating enzyme E2 I (UBE2I), ubiquitin conjugating enzyme E2 L3 (UBE2L3), ribosomal protein L15 (RPL15), chromosome 5 open reading frame 24 (C5ORF24) and FOS-like 2 (FOSL2), anti-GAPDH antibody (Beyotime, China), anti-UBE2I antibody (Proteintech, China), anti-UBE2L3 antibody (Proteintech, China), anti-RPL15 antibody (Proteintech, China), anti-C5ORF24 antibody (Proteintech, China), and anti-FOSL2 antibody (Proteintech, China) were used for immunoblotting.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Virus, Infection, Control, Multiplex sample analysis, Phospho-proteomics