Journal:

Article Title: Multiple activities contribute to Pc2 E3 function

doi: 10.1038/sj.emboj.7600506

Figure Lengend Snippet: Pc2(401–558) colocalizes Ubc9 and CtBP. COS-1 cells were transfected with the indicated expression constructs and visualized by live cell fluorescence microscopy 24 h later. (A) YFP, YFP-Pc2(401–558) or YFP-Pc2 was expressed and cells were stained with Hoechst before visualization. YFP and Hoechst images are shown. (B, C) CFP-CtBP or YFP-Ubc9 was expressed alone (upper panel) or with the indicated Fl-Pc2 constructs (lower panels). CFP and YFP images are shown. (D) CFP-CtBP and YFP-Ubc9 were coexpressed in the presence or absence of Fl-Pc2(401–558). Individual CFP and YFP images along with the merged (overlay) are shown. Scale bar=10 μm.

Article Snippet: Sepharose was washed extensively and bound proteins were analyzed by Western blot with a Ubc9 antibody (Boston Biochem).

Techniques: Transfection, Expressing, Construct, Fluorescence, Microscopy, Staining

Journal:

Article Title: Multiple activities contribute to Pc2 E3 function

doi: 10.1038/sj.emboj.7600506

Figure Lengend Snippet: Pc2(2–288) enhances sumoylation of recruited CtBP. (A) COS-1 cells were cotransfected with T7-CtBP, H6-SUMO1 and Fl-Ubc9 either alone or with the indicated Fl-Pc2 or YFP constructs. Following lysis, proteins were separated by SDS–PAGE and analyzed with a T7 antibody to determine relative levels of CtBP sumoylation. Expression of the Flag and YFP constructs is shown below. (B) COS-1 cells were transfected and analyzed as in panel A, except that Fl-Ubc9 was not present. (C, D) COS-1 cells were transfected with the indicated CtBP, Pc2 and YFP expression constructs. Cell lysates were precipitated with anti-Flag-agarose and analyzed for co-precipitating YFP-PLDLS (C) or T7-CtBP (D). Expression of YFP, Flag and T7 constructs is shown below. The bar denotes the immunoglobulin heavy chain. (E) The expression constructs used in (A–D) are shown schematically (labeling as in Figure 3). (F) T7-CtBP and H6-SUMO1 were coexpressed alone or with Fl-Pc2, Fl-Pc2(401–558), Fl-Pc2(2–288) or a mutant in which cysteine 183 has been altered to alanine. Cell lysates were analyzed by Western blot with a T7 antibody to detect SUMO modified and unmodified CtBP. The positions of molecular weight markers (kDa) are indicated.

Article Snippet: Sepharose was washed extensively and bound proteins were analyzed by Western blot with a Ubc9 antibody (Boston Biochem).

Techniques: Construct, Lysis, SDS Page, Expressing, Transfection, Labeling, Mutagenesis, Western Blot, Modification, Molecular Weight

Journal:

Article Title: Multiple activities contribute to Pc2 E3 function

doi: 10.1038/sj.emboj.7600506

Figure Lengend Snippet: Pc2(2–288) stimulates CtBP sumoylation in vitro. Recombinant T7-CtBP, E1, SUMO1 and Ubc9 (as indicated) were incubated with increasing amounts (1, 5, 10 or 25 ng) of GST-Pc2(2–288)-PIDLRS (A) or GST-Pc2(2–288) (B). In (C), ‘+' indicates 25 ng GST-Pc2(2–288)-PIDLRS or GST-Pc2(2–288). Reactions were terminated by the addition of SDS loading buffer, and SUMO modified CtBP and unmodified CtBP were detected by T7 Western blot. (D) Either GST or GST-Pc2(2–288) bound to glutathione Sepharose was incubated with recombinant Ubc9. Following extensive washing, bound Ubc9 was analyzed by Western blot with a Ubc9 antibody. The input lane is 100 ng Ubc9. (E) Ubc9, E1 and SUMO1 were incubated in vitro for the indicated times with or without GST-Pc2(2–288), and analyzed by nonreducing SDS–PAGE and Western blot with a Ubc9 antibody. The positions of Ubc9 and Ubc9 with SUMO1 attached via a thioester linkage are indicated. The right-hand lanes are reducing SDS–PAGE to disrupt the thioester. (F) SUMO1-loaded E1 and T7-CtBP were incubated with apyrase and the indicated amounts of Pc2(2–288). CtBP and sumoylated CtBP were detected by Western blot with a T7 antibody. The positions of molecular weight markers (kDa) are indicated.

Article Snippet: Sepharose was washed extensively and bound proteins were analyzed by Western blot with a Ubc9 antibody (Boston Biochem).

Techniques: In Vitro, Recombinant, Incubation, Modification, Western Blot, SDS Page, Molecular Weight

Journal:

Article Title: Multiple activities contribute to Pc2 E3 function

doi: 10.1038/sj.emboj.7600506

Figure Lengend Snippet: Analysis of the carboxyl-terminal region of Pc2. (A) COS-1 cells were transfected with Fl-Pc2 or a mutant form in which lysine 492 is altered to arginine, either with or without H6-SUMO1. Lysates were analyzed by Flag Western blot and the unmodified and sumoylated Pc2 are indicated. (B) COS-1 cells were transfected with T7-CtBP, H6-SUMO1 and the indicated Flag-Pc2 constructs. Cell lysates were analyzed by Western blot with a T7 antibody to detect SUMO modified and unmodified CtBP. Expression of Flag constructs is shown below. (C) A series of carboxyl-terminal deletions of Pc2 was tested for their ability to enhance SUMO modification of CtBP in COS-1 cells. (D) COS-1 cells were transfected with the indicated Fl-Pc2 expression constructs and T7-Ubc9. Protein complexes were precipitated with anti-Flag-agarose and analyzed by Western blot for T7-Ubc9. Expression of Ubc9 and the Fl-Pc2 constructs in the lysate was analyzed by direct Western blot. The positions of molecular weight markers (kDa) are indicated. (E) The Pc2 deletion constructs tested in panels C and D are shown schematically (labeling as in Figure 3).

Article Snippet: Sepharose was washed extensively and bound proteins were analyzed by Western blot with a Ubc9 antibody (Boston Biochem).

Techniques: Transfection, Mutagenesis, Western Blot, Construct, Modification, Expressing, Molecular Weight, Labeling

Journal:

Article Title: Multiple activities contribute to Pc2 E3 function

doi: 10.1038/sj.emboj.7600506

Figure Lengend Snippet: A Ubc9 binding domain required for enhancement of CtBP sumoylation. (A) Recombinant T7-CtBP, E1, SUMO1 and Ubc9 (as indicated) were incubated with increasing amounts (1–500 ng) of GST-Pc2(401–558). (B) SUMO1-loaded E1 and T7-CtBP were incubated with apyrase and the indicated amounts of GST-Pc2(401–558) or GST. Reactions were terminated by the addition of SDS loading buffer and SUMO modified and unmodified CtBP were detected by T7 Western blot. (C) COS-1 cells were transfected with T7-CtBP, H6-SUMO1 and the indicated Flag-Pc2 constructs. Cell lysates were analyzed by Western blot with a T7 antibody to detect SUMO modified and unmodified CtBP. (D) The Fl-Pc2 constructs used in (B) were coexpressed with T7-CtBP in COS-1 cells. Lysates were precipitated with anti-Flag-agarose and analyzed for co-precipitating T7-CtBP. The positions of molecular weight markers (kDa) are indicated. (E) The constructs used in panels C and D are shown schematically.

Article Snippet: Sepharose was washed extensively and bound proteins were analyzed by Western blot with a Ubc9 antibody (Boston Biochem).

Techniques: Binding Assay, Recombinant, Incubation, Modification, Western Blot, Transfection, Construct, Molecular Weight

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