Journal: Cell death & disease

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

doi: 10.1038/s41419-024-07271-8

Figure Lengend Snippet: Fig. 1 deSUMO2/3-ylation induces mitophagy and promotes HIM. A Hypoxia induces mitophagy. HeLa cells were transfected with Mito- pHfluorin per 35mm-dish and exposed to normoxia (N) or hypoxia (H;1% O2 for 4, 8, 16, or 24 h) (Scale bar, 10 μm). Upper panel shows that hypoxia-induced mitophagy is detectable as early as 4 h after cells exposed to 1% O2. Histogram in the right panel shows the average number of puncta per cell under indicated time points (n = 19 ~ 51 cells; *p < 0.05; **p < 0.01; ***p < 0.001; ****; unpaired t-test). B, C Hypoxia causes decreased SUMO2/3-ylation (B, n = 5, biological replicates; *p < 0.05; paired t-test) but not SUMO1-ylation (C, n = 6, biological replicates; N.S., non-significant; paired t-test) in HeLa cells. HeLa cells were exposed to 1% O2 for 24 h. Whole cell lysate samples were prepared and blotted as indicated.

Article Snippet: The primary antibodies used were: β-actin (mouse monoclonal Ab; Sigma-Aldrich A2228; 1:20,000 dilution; rabbit polyclonal Ab; ProteinTech 20536-1-AP; 1:5,000 dilution), FEN1 (mouse monoclonal Ab; Santa Cruz biotechnology sc-28355, 1:500 dilution), FIS1 (mouse monoclonal Ab; ProteinTech 66635-1-Ig; 1:1,000 dilution), GAPDH (mouse monoclonal Ab; Santa Cruz biotechnology sc-365062; 1:1,000 dilution), GST (goat polyclonal Ab; GE Healthcare; 1:10,000 dilution), HA-tag (rabbit polyclonal Ab; ProteinTech 66006-2-Ig, 1:2,000 dilution), HIF1α (mouse monoclonal Ab, BD Bioscience 610959; 1:1,000 dilution), His (rabbit polyclonal Ab, Cell Signaling #2365; 1:1,000 dilution), LC3 (rabbit polyclonal Ab, Cell Signaling #4108; 1:2,000 dilution), SENP1 (recombinant monoclonal Ab, Abcam EPR3844, 1:2,000 dilution), SENP3 (rabbit monoclonal Ab, Cell Signaling #5591, 1:10,000 dilution), SUMO1 (rabbit polyclonal Ab, Cell Signaling #4930; 1:1,000 dilution), SUMO2/3 (rabbit monoclonal Ab, Cell Signaling #4971; 1:1,000 dilution), TBC1D17 (rabbit polyclonal Ab, ProteinTech 20482-1-AP; 1:1,000 dilution), and α-Tubulin (rabbit polyclonal Ab, ProteinTech 11224-1-AP; 1:2,000 dilution or mouse monoclonal Ab, ProteinTech 66031-1-Ig; 1:20,000 dilution).

Techniques: Transfection

Journal: Journal of proteome research

Article Title: The human regulatory protein Ki-1/57 is a target of SUMOylation and affects PML nuclear bodies formation

doi: 10.1021/acs.jproteome.7b00001

Figure Lengend Snippet: (A) HEK293T cells were transiently co-transfected with pcDNA-FLAG (empty vector) or pcDNA6-FLAG-Ki-1/57, pcDNA3-Myc-UBC9, and pcDNA3-Myc-SUMO-1. Cells extracts were immunoprecipitated (IP) with G-sepharose beads coupled to antibody against the FLAG tag. The obtained protein complexes were analyzed by immunobloting (IB) as indicated in the panel. Asterisk indicates SUMO-1-conjugated Ki-1/57. (B) HEK293T cells were transiently co-transfected with pcDNA-FLAG (empty vector) or pcDNA6-FLAG-Ki-1/57 or pcDNA6-FLAG-CGI-55, pcDNA3-Myc-UBC9, and pcDNA3-Myc-SUMO-1 or pcDNA3-Myc-SUMO-2. Cells extracts were immunoprecipitated (IP) with G-sepharose beads coupled to anti-FLAG antibody. The obtained protein complexes were analyzed by immunobloting (IB) as indicated in the panel. Asterisk indicates SUMO-2-conjugated Ki-1/57 or SUMO-2-conjugated CGI-55. (C) HEK293T cells were transiently co-transfected with pcDNA-FLAG (empty vector) or pcDNA6-FLAG-CGI-55, pcDNA3-Myc-UBC9, and pcDNA3-Myc-SUMO-1. Cells extracts were immunoprecipitated (IP) with A-sepharose beads coupled to polyclonal rabbit antibody against CGI-55. The obtained protein complexes were analyzed by immunobloting (IB) as indicated in the panel. Asterisk indicates SUMO-1-conjugated CGI-55. (D) HEK293T cells were transiently co-transfected with pcDNA-FLAG (empty vector) or pcDNA6-FLAG-CGI-55 or pcDNA6-FLAG-CGI-55 triple mutant (K3R: K102R, K228R and K281R), pcDNA3-Myc-UBC9, and pcDNA3-Myc-SUMO-1. Cells extracts were immunoprecipitated (IP) with G-sepharose beads coupled to anti-FLAG antibody. The obtained protein complexes were analyzed by immunobloting (IB) as indicated in the panel.

Article Snippet: The eluates were analyzed by Western blot, using rabbit polyclonal anti-SUMO-2 (1:1000 dilution; Abcam), rabbit polyclonal anti-SUMO-1 (1:500 dilution, Abgent) and mouse monoclonal anti-FLAG M2 (1:5000 dilution; Sigma-Aldrich).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, FLAG-tag, Western Blot, Mutagenesis

Journal: Journal of proteome research

Article Title: The human regulatory protein Ki-1/57 is a target of SUMOylation and affects PML nuclear bodies formation

doi: 10.1021/acs.jproteome.7b00001

Figure Lengend Snippet: (A) HEK293T cells were transiently transfected with pcDNA6-FLAG-Ki-1/57 (wt: wild-type) or Ki-1/57 double mutants K336R/K213R or K336R/K276R or triple mutant K336R/K276R/K213R. Cells extracts were immunoprecipitated (IP) with G-sepharose beads coupled to antibody against FLAG tag. The obtained protein complexes were analyzed by western blot using anti-FLAG and anti-SUMO-1 antibodies. Arrow indicates SUMO-1-conjugated Ki-1/57. (B) HEK293T cells were transiently transfected with pcDNA6-FLAG-Ki-1/57 (wt: wild-type) or Ki-1/57 triple mutant K336R/K276R/K213R. Cells extracts were immunoprecipitated (IP) with G-sepharose beads coupled to anti-FLAG antibody. The obtained protein complexes were analyzed by western blot using anti-FLAG and anti-SUMO-2/3 antibodies. Arrow indicates SUMO-2/3-conjugated Ki-1/57. Arrowhead indicates the antibody heavy chain.

Article Snippet: The eluates were analyzed by Western blot, using rabbit polyclonal anti-SUMO-2 (1:1000 dilution; Abcam), rabbit polyclonal anti-SUMO-1 (1:500 dilution, Abgent) and mouse monoclonal anti-FLAG M2 (1:5000 dilution; Sigma-Aldrich).

Techniques: Transfection, Mutagenesis, Immunoprecipitation, FLAG-tag, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: SUMO2 modification of Aurora B and its impact on follicular development and atresia in the mouse ovary

doi: 10.3892/ijmm.2018.3541

Figure Lengend Snippet: Identification of SUMOylation of Aurora B in vivo and in vitro . (A) Lysates of granulosa cells were immunoprecipitated with IGG, SUMO1 and anti-SUMO2 antibodies, prior to immunoblotting with anti-Aurora B antibody. The arrow represents the complex protein of Aurora B-SUMO2. On the left, the input was immunoblotted with anti-Aurora B antibody and anti-β-actin antibody, respectively. (B) The SUMOylation site was predicted by SUMOsp2.0 to be at Lys-207 in Mus musculus . (C and D) The co-transfection of HA-Aurora B or HA-Aurora B K207R with Flag-SUMO2, followed by immunoprecipitation with the anti-Flag antibody and immunoblotting with the anti-HA antibody. The arrow represents the complex protein of Aurora B-SUMO2, and the intensity of the bands was analyzed using ImageJ software. (E and F) Co-transfection of HA-Aurora B or HA-Aurora B K207R with Flag-SUMO2, followed by immuno-precipitation with the anti-HA antibody and immunoblotting with the anti-Flag antibody. The arrow represents the complex protein of Aurora B-SUMO2, and the intensity of the bands was analyzed using ImageJ software. Each experiment was repeated three times, and the values are presented as the mean ± standard deviation. *** P<0.001. SUMO1, anti-small ubiquitin-related modifier 1; IP, immunoprecipitate; IB, immunoblot.

Article Snippet: The granulosa cells were washed with ice-cold PBS and lysed on ice in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% Triton X-100] that was supplemented with 1% PMSF, 1% cocktail and 20 mM NEM for 20 min; the protein samples were incubated with 1 μ g of the respective primary antibodies overnight at 4°C, a polyclonal rabbit anti-SUMO1 antibody (10329-1-AP) and a polyclonal rabbit anti-SUMO2 antibody (11251-1-AP) (both from Proteintech, as described above), a monoclonal mouse anti-HA antibody (M180-3; Medical and Biological Laboratories, Co., Ltd.), and a polyclonal rabbit anti-Flag antibody (20543-1-AP; Proteintech), and subsequently 40 μ l protein A+G Agarose (Beyotime Biotechnology Co., Ltd., Shanghai, China) was added for 2 h at 4°C.

Techniques: In Vivo, In Vitro, Immunoprecipitation, Western Blot, Cotransfection, Software, Standard Deviation, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A) In situ hybridization analyses of SUMO pathway components at different developmental stages in the mouse (from 8.0 to 11.5 dpc, with ventral view at 8.0 dpc, and lateral view 9.5–11.5 dpc). Transcripts were seen in most embryonic regions, but enhanced expression could be noted in sites of extensive morphogenesis, such as the neural folds (8.0 dpc), branchial arches and limb buds (9.5 to 11.5 dpc). Arrows indicate cardiogenic regions at 8.0 dpc. (B) In situ hybridization revealed cardiac expression of SUMO-1 and SUMO-2, with stronger expression in the outer curvature, as demonstrated by Nppa transcripts. la – left atria; lv – left ventricle; rv – right ventricle; ra – right atria; oft – outflow tract.

Article Snippet: Cells were then incubated with primary rabbit polyclonal anti-Nkx2-5 antibody (1∶200 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-SUMO-1 (1∶100 dilution, Zymed Laboratories), and Alexa 555 anti-rabbit secondary antibody (1∶200 dilution, Invitrogen), and were mounted in Vectashield/DAPI (Vector Laboratories).

Techniques: In Situ Hybridization, Expressing

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A) Transiently transfected HEK293T cells expressing Nkx2-5 and HA-SUMO-1 proteins alone or simultaneously. Addition of HA-SUMO-1 led to the appearance of two extra bands by western blot, specific for both HA and Nkx2-5 antibodies. (B) Co-IP experiments performed in cardiac HL-1 cells with Nkx2-5 antibody display a similar pattern of SUMOylation (arrowheads) but with increased detection of the slow-migrating SUMOylated Nkx2-5 band. Stars (*) indicate SUMOylated proteins co-precipitated with Nkx2-5 antibodies. (C) Incubation of cellular extracts with λPPA caused the disappearance of the slower migrating band detected by Nkx2-5 antibodies (lower left panel), while no change in the pattern of migration of the Nkx2-5/SUMO-1 co-stained bands was observed (upper left panel).

Article Snippet: Cells were then incubated with primary rabbit polyclonal anti-Nkx2-5 antibody (1∶200 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-SUMO-1 (1∶100 dilution, Zymed Laboratories), and Alexa 555 anti-rabbit secondary antibody (1∶200 dilution, Invitrogen), and were mounted in Vectashield/DAPI (Vector Laboratories).

Techniques: Transfection, Expressing, Western Blot, Co-Immunoprecipitation Assay, Incubation, Migration, Staining

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A) Nkx2-5 sequences from fish to humans were analyzed bioinformatically and canonical conserved sites were identified at the amino-terminal region of the protein. (B–C) HEK293T cells were co-transfected with HA-SUMO-1 and single putative SUMOylation site mutants (K51R, K103R, K109R or the triple mutant 3K–R), and the band pattern was assessed by western blot with antibodies for HA and Nkx2-5. Transfection of K51R caused the disappearance of one previously detected SUMOylated band, indicating that K51 is a site for SUMO modification. All other mutants had identical SUMOylation pattern to the wild-type protein, suggesting that those were not SUMOylated sites. TND, TN domain; HD, homeodomain; NKD, NK2 specific domain; YRD, tyrosine-rich domain.

Article Snippet: Cells were then incubated with primary rabbit polyclonal anti-Nkx2-5 antibody (1∶200 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-SUMO-1 (1∶100 dilution, Zymed Laboratories), and Alexa 555 anti-rabbit secondary antibody (1∶200 dilution, Invitrogen), and were mounted in Vectashield/DAPI (Vector Laboratories).

Techniques: Transfection, Mutagenesis, Western Blot, Modification

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A) HEK293T cells were transiently transfected with Nkx2-5, HA-SUMO-1 and the Nppa promoter. Activity is expressed as the fold-increase in luciferase expression. (B) No significant SUMO-1-mediated activation of the Nppa promoter was observed for other cardiac transcriptions factors (Tbx20a and GATA4/5), indicating that this effect was specific to Nkx2-5. (C) HEK293T were transiently transfected with wildtype Nkx2-5 or with a homeodomain point mutant that decreases DNA binding affinity. Addition of mutant protein abolishes SUMO-mediated activation. (D) The Drosophila homolog of Nkx2-5 gene, tinman , also displays SUMO-1-dependent transcriptional activity.

Article Snippet: Cells were then incubated with primary rabbit polyclonal anti-Nkx2-5 antibody (1∶200 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-SUMO-1 (1∶100 dilution, Zymed Laboratories), and Alexa 555 anti-rabbit secondary antibody (1∶200 dilution, Invitrogen), and were mounted in Vectashield/DAPI (Vector Laboratories).

Techniques: Transfection, Activity Assay, Luciferase, Expressing, Activation Assay, Mutagenesis, Binding Assay

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A–C) SUMO-1 and Nkx2-5 failed to activate the cardiac promoters Gja5 (A) and Pitx2 (B) but could weakly activate Isl1 (B) and SM22 (C) promoters in HEK293T cells. (D) qPCR from First Heart Field (FHF) and trunk region enriched for Second Heart Field progenitors (eSHF) show presence of several SUMO components in both regions analysed. Regions used in this experimented are represented as red lines on E8.5 mouse embryo diagram (left). The relative levels on both regions were shown by non-saturated cycling PCR (30 cycles) followed by gel electrophoresis. (E) Immunohistochemistry of E9.5 embryos sections show Sumo-1 and Sumo-2 widely expressed and this pattern overlaps with Nkx2-5 expressing regions derived from Second Heart Field (SHF), including the outflow tract (oft) and lateral mesoderm (lm). lm – lateral mesoderm; oft – outflow tract; fg – foregut.

Article Snippet: Cells were then incubated with primary rabbit polyclonal anti-Nkx2-5 antibody (1∶200 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-SUMO-1 (1∶100 dilution, Zymed Laboratories), and Alexa 555 anti-rabbit secondary antibody (1∶200 dilution, Invitrogen), and were mounted in Vectashield/DAPI (Vector Laboratories).

Techniques: Nucleic Acid Electrophoresis, Immunohistochemistry, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A) SUMO-1 stimulates Nkx2-5-driven reporter activity in a dose-dependent manner, but SUMOylation-defective mutant proteins K51R and 3K-R had no effect on SUMO-dependent activation. Nkx2-5 and HA-SUMO1 protein levels were detected in cell extracts by western blot, with α-tubulin as loading control. (B) Immunofluorescence of transiently transfected HEK293T cells show normal nuclear localization of Nkx2-5 mutants K51R, K103R, K109R and 3K–R when compared to wildtype protein. (C) Nkx2-5 K51R binds to the NKE site with similar affinities to wildtype protein in HEK293T cells using EMSA. Overexpression of SUMO-1 leads to no detectable change in DNA affinities. S, specific oligonucleotide; NS, non-specific oligonucleotide; SS, supershift. Note that western blots were performed using extracts for Luciferase readings without NEM, therefore only the non-SUMOylated form of Nkx2-5 was detected.

Article Snippet: Cells were then incubated with primary rabbit polyclonal anti-Nkx2-5 antibody (1∶200 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-SUMO-1 (1∶100 dilution, Zymed Laboratories), and Alexa 555 anti-rabbit secondary antibody (1∶200 dilution, Invitrogen), and were mounted in Vectashield/DAPI (Vector Laboratories).

Techniques: Activity Assay, Mutagenesis, Activation Assay, Western Blot, Immunofluorescence, Transfection, Over Expression, Luciferase

Journal: PLoS ONE

Article Title: Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

doi: 10.1371/journal.pone.0024812

Figure Lengend Snippet: (A–B) Mutagenesis of every lysine in Nkx2-5 in conjunction with K51R failed to change its SUMOylation pattern, as detected by western blot. (B) Most double mutant proteins were not capable of attenuating the activation exerted by SUMO-1 on Nkx2-5 when the Nppa promoter was used in co-transfection experiments performed in HEK293T cells. The exception was the double mutant K51/191R mutant that showed strong activation. (C) Deletion of the carboxi-terminal region of Nkx2-5 (ΔC) impaired SUMO-mediated activation on the Nppa promoter, despite enhancing Nkx2-5 stability in transient assays (inset). (D) A SUMO-1/Nkx2-5 fusion construct failed to elicit any activation of the Nppa promoter when compared to the Nkx2-5 WT construct alone, but activation was restored upon addition of exogenous HA-SUMO-1 constructs. Inset: western blot of extracts used for luciferase readings show presence of exogenously expressed Nkx-2-5 WT, HA-SUMO-1 and SUMO-1/Nkx2-5 fusions (SUMO-1∼Nkx2-5) WT and K51R. Endogenous SUMO-1 and possibly RanGAP1 (*) are also detected.

Article Snippet: Cells were then incubated with primary rabbit polyclonal anti-Nkx2-5 antibody (1∶200 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-SUMO-1 (1∶100 dilution, Zymed Laboratories), and Alexa 555 anti-rabbit secondary antibody (1∶200 dilution, Invitrogen), and were mounted in Vectashield/DAPI (Vector Laboratories).

Techniques: Mutagenesis, Western Blot, Activation Assay, Cotransfection, Construct, Luciferase