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

Journal: Plant Physiology

Article Title: Defining the SUMO System in Maize: SUMOylation Is Up-Regulated during Endosperm Development and Rapidly Induced by Stress ^{1 [OPEN]}

doi: 10.1104/pp.16.00353

Figure Lengend Snippet: A recombinant SUMO conjugation system generated with maize components. Various combinations of SUMO1a, the SAE1 and SAE2a polypeptides of the E1 heterodimer, and the SCE1b and SCE1f E2 enzymes bearing 6His, HA, FLAG, and Myc epitopes were expressed in E. coli. Crude extracts prepared after an 8-h induction at 30°C were probed with antibodies against AtSUMO1 (6His-SUMO1a), HA (HA-SAE1), FLAG (SAE2a-FLAG), and Myc (SCE1-Myc; right gel) or anti-6His antibodies (6His-SUMO1a; left gel). SCE1(C-S) indicates the Ser substitutions of the active-site Cys. A, The E1 and E2 enzymes are sufficient to drive SUMOylation. SUMO1a and the SAE1/2 heterodimer were coexpressed with wild-type or C-S versions of SCE1b (left gels) and an E. coli-optimized version of SCE1f (right gels). B, The truncated SAE2a splice variant T2 (Trunc) is functional but less active than full-length SAE2a (FL). The SAE2a polypeptides bearing a C-terminal FLAG tag were coexpressed individually with SUMO1a, SAE1, and SCE1b. Asterisk locates the T2 SAE2a truncation. C, Direct comparison of the conjugating activity of the class II E2 SCE1f with representative class I E2s, SCE1b, and SCE1d. D, Both class I and class II SCE1s catalyze the formation of poly-SUMO chains. Wild-type SUMO1a (WT) or the Lys-less K0 mutant blocked in forming SUMO chains were coexpressed with the SAE1/2 heterodimer and either SCE1b or SCE1f.

Article Snippet: Analogous to ubiquitylation, SUMOylation is driven by an ATP-dependent E1→E2→E3 conjugation cascade sequentially involving a heterodimeric SUMO-activating enzyme (SAE1/2), a single SUMO-conjugating enzyme (SCE), and a small collection of SUMO protein ligases ( Geiss-Friedlander and Melchior, 2007 ; Jentsch and Psakhye, 2013 ).

Techniques: Recombinant, Conjugation Assay, Generated, Variant Assay, Functional Assay, FLAG-tag, Comparison, Activity Assay, Mutagenesis