human anti sumo1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: ALS driven by mutant NEK1 aggregation is accelerated by Pml loss, but clinically reversed through pharmacologic induction of Pml -mediated degradation

doi: 10.1101/2024.11.23.622051

Figure Lengend Snippet: Therapeutic benefit of Pml induction by IFNα or poly(I:C) through NEK1 t clearance in vivo . A) NEK1 t modification by SUMO1 and SUMO2/3. HEK293 cells were transfected with the indicated expression vectors and treated or not with IFNα. IP: immuno-precipitation, WCL: whole cell lysate. B) IFNα treatment induces proteasome-dependent degradation of NEK1 t in HEK293 cells. MG132: proteasome inhibitor, n=3. C) PML silencing impedes IFNα-induced NEK1 t degradation. Quantifications (right), n=3. D) Western blot of NEK1 t protein from the spinal cord of age-matched Nek1 t/t or Nek1 t/t Pml −/− mice following IFNα or poly(I:C) therapy, (*) non-specific band. E) IFNα or poly(I:C) therapy eliminates NEK1 t aggregates in αMNs of Nek1 t/t mice. Quantifications: ≥30 αMNs from ≥3 mice F) IFNα or poly(I:C) therapies selectively extend lifespan in Pml -proficient Nek1 t/t mice. Arrowhead: therapy initiation, continued until death. G) IFNα or poly(I:C) therapies improve muscle strength and neuromuscular function selectively in Pml -proficient Nek1 t/t ALS mice. Trendlines in Supplementary Fig.S2E.

Article Snippet: The following antibodies were used for Western blots in HEK293 or Neuro2A cells: anti-GST (Cell signaling, Cat # 2624S), human anti-GAPDH (Santa Cruz, Cat # sc-32233;), human anti-actin (BioLegend, Cat # 622102), human anti-YY1 (Santa Cruz, H-10, Cat # sc-7341), human anti-PML (Santa Cruz, H-238, Cat # sc-5621 and Santa Cruz, E-11, Cat # sc-377390), human anti-SUMO1 (CST, Cat # 4930), human anti-SUMO2/3 (Abcam, Cat # ab3742).

Techniques: In Vivo, Modification, Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Real-time RT-PCR and Western blots for SUMO1 with and without SUMO1 siRNA transfection in fibroblasts . Three SSc fibroblast strains (two with anti-topo I and one with anti-RNA polymerase III positive serum) were transfected with SUMO1 siRNA. After 48-hour transfection, total RNAs were used for measuring SUMO1 transcript levels (Figure 4a), and the nuclear extracts were used for measuring SUMO1 protein (Figure 4b). Error bars indicate standard deviation.

Article Snippet: Resolved proteins were transferred onto nitrocellulose membranes and incubated with 1:1,000 diluted primary antibodies including mouse anti-human topo I (ImmunoVision, Springdale, AR, USA), anti-human SUMO1 (ABGENT, San Diego, CA, USA) and anti-collagen type I, individually.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Standard Deviation

Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Catalytic function of topo I in cultured SSc fibroblasts with and without SUMO1 siRNA transfection . A serial dilution of the nuclear extract containing topo I obtained from SSc fibroblasts was used to relax 0.25 μg supercoiled DNA. In this figure, the supercoiled DNA band was completely transformed to relaxed DNA at dilutions of one half and one in the fibroblasts without siRNA transfection or non-target siRNA transfection. In contrast, this change was observed between the one-eighth and one-fourth dilutions in the fibroblasts with SUMO1 transfection, which indicates a higher efficiency of catalytic function of topo I after SUMO1 inhibition in the fibroblasts. According to the intensity of the bands of remaining supercoiled DNA in serial dilutions in the assays of three fibroblast strains, these changes are significant. The P- values are 0.045 and 0.027 at the one-fourth dilution for comparisons between SUMO1 siRNA vs. non-target siRNA, or vs. without siRNA transfected fibroblasts, respectively (Student's t -test). This is representative of three SSc fibroblast strains examined in SUMO1 siRNA studies. * A , supercoiled DNA; B , relaxed DNA.

Article Snippet: Resolved proteins were transferred onto nitrocellulose membranes and incubated with 1:1,000 diluted primary antibodies including mouse anti-human topo I (ImmunoVision, Springdale, AR, USA), anti-human SUMO1 (ABGENT, San Diego, CA, USA) and anti-collagen type I, individually.

Techniques: Cell Culture, Transfection, Serial Dilution, Transformation Assay, Inhibition

Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Western blots show sumoylation of recombinant human topo I . Recombinant human topo I protein was subjected to the sumoylation reaction and examined by Western blotting using anti-topo I (I) and anti-SUMO1 antibodies (II). Compared to topo I protein without sumoylation reaction (topo I A ), topo I protein with sumoylation reaction (topo I B ) showed poly-sumoylation of topo I (II). The assays showed similar results in triplicates.

Article Snippet: Resolved proteins were transferred onto nitrocellulose membranes and incubated with 1:1,000 diluted primary antibodies including mouse anti-human topo I (ImmunoVision, Springdale, AR, USA), anti-human SUMO1 (ABGENT, San Diego, CA, USA) and anti-collagen type I, individually.

Techniques: Western Blot, Recombinant

Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Measurement of catalytic function of recombinant human topo I with and without sumoylation reaction . Recombinant human topo I proteins were sumoylated with either mutant sumo1 or wild type sumo1 or negative control (without sumoylation), and then were examined for their catalytic function in a serial dilution. Sumoylation of topo I with wild type sumo1 showed a reduction of efficiency in catalytic function (supercoiled DNA disappeared at the dilution of topo I concentration of 30) compared to the topo I protein sumoylated with mutant sumo1 or negative control (supercoiled DNA disappeared at topo I concentration of 22.5). This is representative of three assays. * A , standard supercoiled DNA band; B , standard relaxed DNA bands.

Article Snippet: Resolved proteins were transferred onto nitrocellulose membranes and incubated with 1:1,000 diluted primary antibodies including mouse anti-human topo I (ImmunoVision, Springdale, AR, USA), anti-human SUMO1 (ABGENT, San Diego, CA, USA) and anti-collagen type I, individually.

Techniques: Recombinant, Mutagenesis, Negative Control, Serial Dilution, Concentration Assay

Journal: PLoS ONE

Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

doi: 10.1371/journal.pone.0033115

Figure Lengend Snippet: A) Representative confocal micrographs showing the nuclear and cytoplasmatic expression of human POLR2E and SUMO-1 proteins in vivo, in kidney sections of cisplatin-AKI (AKI-CIS) mice treated or not with MVs and sacrificed 48 hours later, and in vitro by TECs treated with cisplatin and cultured in the absence (vehicle) or in the presence of 50 µg of MVs (MV) for 24 hours. Nuclei were counterstained with Hoechst dye. Original magnification: ×400 for kidney sections and ×630 for TECs. B) 1×10 5 TECs treated with cisplatin and cultured in the absence (CIS) or in the presence of two different preparations of MVs (+MV) for 1 hour were analysed by RT-PCR for specific human mRNA POLR2E . Bands of PCR products specific for human POLR2E of the expected size (90 pb) were detected in a 4% agarose gel electrophoresis. As positive control the extract of human bone marrow-derived MSCs (BM-MSC) was used. The * indicates the control without cDNA.

Article Snippet: The following antibodies were used: rabbit anti-human POLR2E (Abcam) and rabbit anti human SUMO-1 (Abcam).

Techniques: Expressing, In Vivo, In Vitro, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Derivative Assay

Journal: PLoS ONE

Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

doi: 10.1371/journal.pone.0033115

Figure Lengend Snippet: A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of SUMO-1 , POLR2 and Act B transcripts in MVs treated or not with RNase, express as 2 -δCt , as described in material and methods.

Article Snippet: The following antibodies were used: rabbit anti-human POLR2E (Abcam) and rabbit anti human SUMO-1 (Abcam).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

doi: 10.1371/journal.pone.0033115

Figure Lengend Snippet: A) Representative confocal micrographs showing the nuclear and cytoplasmatic expression of human POLR2E and SUMO-1 proteins in vivo, in kidney sections of cisplatin-AKI (AKI-CIS) mice treated or not with MVs and sacrificed 48 hours later, and in vitro by TECs treated with cisplatin and cultured in the absence (vehicle) or in the presence of 50 µg of MVs (MV) for 24 hours. Nuclei were counterstained with Hoechst dye. Original magnification: ×400 for kidney sections and ×630 for TECs. B) 1×10 5 TECs treated with cisplatin and cultured in the absence (CIS) or in the presence of two different preparations of MVs (+MV) for 1 hour were analysed by RT-PCR for specific human mRNA POLR2E . Bands of PCR products specific for human POLR2E of the expected size (90 pb) were detected in a 4% agarose gel electrophoresis. As positive control the extract of human bone marrow-derived MSCs (BM-MSC) was used. The * indicates the control without cDNA.

Article Snippet: Section were blocked and labelled with rabbit anti-human POLR2E (Abcam, Cambridge Science Park, UK) (1∶300 dilution) or rabbit anti-human SUMO-1 (Abcam) (1∶300 dilution).

Techniques: Expressing, In Vivo, In Vitro, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Derivative Assay

Journal: PLoS ONE

Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

doi: 10.1371/journal.pone.0033115

Figure Lengend Snippet: A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of SUMO-1 , POLR2 and Act B transcripts in MVs treated or not with RNase, express as 2 -δCt , as described in material and methods.

Article Snippet: Section were blocked and labelled with rabbit anti-human POLR2E (Abcam, Cambridge Science Park, UK) (1∶300 dilution) or rabbit anti-human SUMO-1 (Abcam) (1∶300 dilution).

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: Identifications of SUMO-1 cDNA and Its Expression Patterns in Pacific White Shrimp Litopeanaeus vannamei

doi:

Figure Lengend Snippet: Nucleotide and deduced amino acid sequences of L. vannamei SUMO-1 (LvSUMO-1) cDNA. Amino acids are indicated as single capital letters under each triplet codon of the nucleotide sequence. The start codon is underlined and an asterisk (*) indicates the stop codon. Sumo modification prediction site on the polypeptide chain was showed in capital letters (AKPE). The polyadenylation signal (ATTAAA) is typed in capital letters underlined.

Article Snippet: Proteins were transferred onto PVDF membrane (Bio Rad) in electoblotting buffer (25mM Tris-HCl, 190mM Glycine, 20% methanol) at a constant current of 2.5mA for 2 h. The membrane was immersed in blocking buffer (5% nonfat dry milk, 1X Tris buffer saline (TBS), pH 7.4, 0.1% Tween 20) at room temperature for 1 hr followed by incubation with rabbit polyclonal antibody to human SUMO-1 (c-terminal) (Abgent) in antibody dilution buffer (1% nonfat dry milk, 1x Tris buffer saline(TBS), pH 7.4, 0.1% Tween 20) at a concentration of 1:500 at 4 C overnight.

Techniques: Sequencing, Modification

Journal: International Journal of Biological Sciences

Article Title: Identifications of SUMO-1 cDNA and Its Expression Patterns in Pacific White Shrimp Litopeanaeus vannamei

doi:

Figure Lengend Snippet: Amino acid sequence alignment among different SUMO-1 proteins. The amino acid sequences of Human (Accession no. AAH53528), Xenopus (Accession no. Q6DEP7), Zebrafish (Accession no. Q7SZR5), Brine shrimp: (Accession no. ABQ41279), Honeybee (Accession no. XP_392826), Wesp (Accession no. XP_001607301), Nematode (Accession no. CAP33470) and yeast (Accession no. NP_010798) are aligned. C-terminal diglycine motif, a site used for propeptide processing, is boxed, and the predicted cleavage site is marked by an arrowhead. A glutamine residue known to be important for the interaction of human SUMO-1 with protease Senp2 is asterisked.

Article Snippet: Proteins were transferred onto PVDF membrane (Bio Rad) in electoblotting buffer (25mM Tris-HCl, 190mM Glycine, 20% methanol) at a constant current of 2.5mA for 2 h. The membrane was immersed in blocking buffer (5% nonfat dry milk, 1X Tris buffer saline (TBS), pH 7.4, 0.1% Tween 20) at room temperature for 1 hr followed by incubation with rabbit polyclonal antibody to human SUMO-1 (c-terminal) (Abgent) in antibody dilution buffer (1% nonfat dry milk, 1x Tris buffer saline(TBS), pH 7.4, 0.1% Tween 20) at a concentration of 1:500 at 4 C overnight.

Techniques: Sequencing

Journal: International Journal of Biological Sciences

Article Title: Identifications of SUMO-1 cDNA and Its Expression Patterns in Pacific White Shrimp Litopeanaeus vannamei

doi:

Figure Lengend Snippet: Alignment of the LvSUMO-1 (sh1) with those of human SUMO-1 (hu1: GenBank Accession No. NP_003343), human SUMO-2(hu2: GenBank Accession No. P61956), human SUMO-3(hu: GenBank Accession No. NP_008867), mouse SUMO-1(m1: GenBank Accession No. NP_033486), mouse SUMO-2(m2: GenBank Accession No. NP_579932), mouse SUMO-3(m3: GenBank Accession No. Q9Z172) and Drosophilar smt3(dro: GenBank Accession No. NP_477411). Identical residues among all sequences are shown in black background and gaps are shown by hyphens. vanSUMO-1 grouped with human and mouse SUMO-1, is boxed.

Article Snippet: Proteins were transferred onto PVDF membrane (Bio Rad) in electoblotting buffer (25mM Tris-HCl, 190mM Glycine, 20% methanol) at a constant current of 2.5mA for 2 h. The membrane was immersed in blocking buffer (5% nonfat dry milk, 1X Tris buffer saline (TBS), pH 7.4, 0.1% Tween 20) at room temperature for 1 hr followed by incubation with rabbit polyclonal antibody to human SUMO-1 (c-terminal) (Abgent) in antibody dilution buffer (1% nonfat dry milk, 1x Tris buffer saline(TBS), pH 7.4, 0.1% Tween 20) at a concentration of 1:500 at 4 C overnight.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: Identifications of SUMO-1 cDNA and Its Expression Patterns in Pacific White Shrimp Litopeanaeus vannamei

doi:

Figure Lengend Snippet: Western Blot analysis of shrimp SUMO-1 transcripts from various tissues compost of heart(H), intestine(I), hepatopancrease(Hep), haemocytes(Cy), lymphoid(L), brain and nerve(BN), Gill(G), pleopods(P) and abdominal muscle(AM) in cytosolic and nuclear protein fraction(M=protein marker). Protein expression level of shrimp SUMO-1 was very high expressed in hepatopancreas both in cytosilic and nuclear protein fraction. In addition, the expression in were found in heart and intestine of cytoplasmic protein fraction and in intestine and lymphoid of nuclear protein fraction.

Article Snippet: Proteins were transferred onto PVDF membrane (Bio Rad) in electoblotting buffer (25mM Tris-HCl, 190mM Glycine, 20% methanol) at a constant current of 2.5mA for 2 h. The membrane was immersed in blocking buffer (5% nonfat dry milk, 1X Tris buffer saline (TBS), pH 7.4, 0.1% Tween 20) at room temperature for 1 hr followed by incubation with rabbit polyclonal antibody to human SUMO-1 (c-terminal) (Abgent) in antibody dilution buffer (1% nonfat dry milk, 1x Tris buffer saline(TBS), pH 7.4, 0.1% Tween 20) at a concentration of 1:500 at 4 C overnight.

Techniques: Western Blot, Marker, Expressing

Journal: International Journal of Biological Sciences

Article Title: Identifications of SUMO-1 cDNA and Its Expression Patterns in Pacific White Shrimp Litopeanaeus vannamei

doi:

Figure Lengend Snippet: Differentiation stage of shrimp SUMO-1 gene expression by RT-PCR (Nau=nauplius, PL1= postlarvae 1 day, PL10= postlarvae 10 days, PL19= postlarvae 19 day and Adu= adult). The expression level is up regulated in all postlavae stated and adult.

Article Snippet: Proteins were transferred onto PVDF membrane (Bio Rad) in electoblotting buffer (25mM Tris-HCl, 190mM Glycine, 20% methanol) at a constant current of 2.5mA for 2 h. The membrane was immersed in blocking buffer (5% nonfat dry milk, 1X Tris buffer saline (TBS), pH 7.4, 0.1% Tween 20) at room temperature for 1 hr followed by incubation with rabbit polyclonal antibody to human SUMO-1 (c-terminal) (Abgent) in antibody dilution buffer (1% nonfat dry milk, 1x Tris buffer saline(TBS), pH 7.4, 0.1% Tween 20) at a concentration of 1:500 at 4 C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Biological Sciences

Article Title: Identifications of SUMO-1 cDNA and Its Expression Patterns in Pacific White Shrimp Litopeanaeus vannamei

doi:

Figure Lengend Snippet: Shrimp SUMO-1 gene expression in different molting stage by RT-PCR. (A= postmolt, B=intermolt, C=premolt). SUMO-1 mRNA level is low expressed between postmolt and intermolt but high expressed in premolt.

Article Snippet: Proteins were transferred onto PVDF membrane (Bio Rad) in electoblotting buffer (25mM Tris-HCl, 190mM Glycine, 20% methanol) at a constant current of 2.5mA for 2 h. The membrane was immersed in blocking buffer (5% nonfat dry milk, 1X Tris buffer saline (TBS), pH 7.4, 0.1% Tween 20) at room temperature for 1 hr followed by incubation with rabbit polyclonal antibody to human SUMO-1 (c-terminal) (Abgent) in antibody dilution buffer (1% nonfat dry milk, 1x Tris buffer saline(TBS), pH 7.4, 0.1% Tween 20) at a concentration of 1:500 at 4 C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: Human immunodeficiency virus type 1 impairs sumoylation

doi: 10.26508/lsa.202101103

Figure Lengend Snippet: (A, B) Assessment of EGFP, HIV-1 Rev, and HIV-1 integrase expression by Western blot in HEK293 cells (A), or in CD4 + Jurkat T lymphocytes (B), transfected or electroporated with the lentiviral vector pfNL43-dE-EGFP encoding HIV-1. The EGFP cassette was inserted within the HIV-1 env gene. (C) HEK293 cells or CD4 + Jurkat T lymphocytes were transfected or electroporated with a vector encoding EGFP only. Global SUMO1 and SUMO2/3 conjugates were analyzed by Western blot. (D) HeLa cells were transfected with the lentiviral vector pfNL43-dE-EGFP encoding HIV-1, and lysed 72 h post-transfection to assess global sumoylation levels, as well as UBA2, EGFP, HIV-1 Rev, and HIV-1 integrase expression by Western blot. Densitometric quantifications of SUMO and UBA2 signals were normalized to GAPDH expression (which serves as a loading control). Data are presented as mean ± SEM (n = 3), P -values were calculated using t test assuming unequal variances. Control: cells transfected with an empty vector backbone devoid of HIV-1 genes, HIV: cells expressing the HIV-1 genome.

Article Snippet: Human anti-SUMO1 (4930S) and anti-SUMO2/3 (4971S) antibodies were purchased from Cell Signaling Technology and human anti-actin (622102) antibody was purchased from BioLegend.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: Life Science Alliance

Article Title: Human immunodeficiency virus type 1 impairs sumoylation

doi: 10.26508/lsa.202101103

Figure Lengend Snippet: (A, B) A lentiviral vector (pfNL43-dE-EGFP) encoding HIV-1 (containing all nine viral genes: gag , pol , tat , rev , vif , vpr , vpu , nef , and env ) was transfected into HEK293 cells (A) or electroporated to CD4 + Jurkat T lymphocytes (B), allowing reverse transcription and integration, followed by viral protein expression in cells. Neither the envelope protein nor any viral particles are produced because of the insertion of an EGFP cassette within the env gene. Expression levels of EGFP and two viral proteins, Rev and integrase, are shown in . This system allows in vitro simulation of HIV-1 infection in a safe, reproducible and quantifiable manner. Cells were lysed at indicated times post-transfection, and global sumoylation levels were assessed by Western blot, using human anti-SUMO1 and anti-SUMO2/3 antibodies. Representative blots are shown. Densitometric quantifications of SUMO signals were normalized to actin expression (which serves as a loading control). Data are presented as mean ± SEM (n = 4 for HEK293 cells, n = 7 for Jurkat cells), asterisks denote statistical significance ( P -values were calculated using t test assuming unequal variances, ns, not significant). Control: cells transfected with an empty vector backbone devoid of HIV-1 genes, HIV, cells expressing the HIV-1 genome.

Article Snippet: Human anti-SUMO1 (4930S) and anti-SUMO2/3 (4971S) antibodies were purchased from Cell Signaling Technology and human anti-actin (622102) antibody was purchased from BioLegend.

Techniques: Plasmid Preparation, Transfection, Expressing, Produced, In Vitro, Infection, Western Blot

Journal: Life Science Alliance

Article Title: Human immunodeficiency virus type 1 impairs sumoylation

doi: 10.26508/lsa.202101103

Figure Lengend Snippet: (A, B) HEK293 cells (A) or CD4 + Jurkat T lymphocytes (B) were transfected or electroporated with the lentiviral vector pfNL43-dE-EGFP encoding the HIV-1 genome as described above, then treated for 24 h with 3 μM MG132, a proteasome inhibitor drug, before lysis (at indicated times post-transfection/electroporation). Global SUMO1 and SUMO2/3 conjugates were analyzed by Western blot. Densitometric quantifications of SUMO signals were normalized to actin expression (which serves as a loading control). Data are presented as mean ± SEM (n = 5 for HEK293 cells, n = 4 for Jurkat cells), asterisks denote statistical significance ( P -values were calculated using t test assuming unequal variances, ns: not significant). Control: cells transfected with an empty vector backbone devoid of HIV-1 genes, HIV: cells expressing the HIV-1 genome. Ubiquitin blots are shown in .

Article Snippet: Human anti-SUMO1 (4930S) and anti-SUMO2/3 (4971S) antibodies were purchased from Cell Signaling Technology and human anti-actin (622102) antibody was purchased from BioLegend.

Techniques: Transfection, Plasmid Preparation, Lysis, Electroporation, Western Blot, Expressing

Journal: Life Science Alliance

Article Title: Human immunodeficiency virus type 1 impairs sumoylation

doi: 10.26508/lsa.202101103

Figure Lengend Snippet: (A) HEK293 cells or CD4 + Jurkat T lymphocytes were transfected or electroporated with the lentiviral vector pfNL43-dE-EGFP encoding HIV-1 as described above, followed by lysis at indicated times post-transfection to assess the level of free (unconjugated) SUMO1 and SUMO2/3 proteins by Western blot. (B) Western blots show UBA2, SAE1 and UBC9 protein levels in HEK293 and Jurkat cells. Cells were lysed and proteins were analyzed at indicated times after transfection or electroporation with HIV-1. HIV-1 specifically down-regulates the host UBA2 protein, a subunit of the heterodimeric SUMO E1 enzyme. Densitometric quantifications of UBA2, SAE1, and UBC9 signals were normalized to tubulin expression, which serves as a loading control. Data are presented as mean ± SEM (n ≥ 3 for HEK293 and Jurkat cells), asterisks denote statistical significance ( P -values were calculated using t test assuming unequal variances, ns, not significant). Control, cells transfected with an empty vector backbone devoid of HIV-1 genes; HIV, cells expressing the HIV-1 genome.

Article Snippet: Human anti-SUMO1 (4930S) and anti-SUMO2/3 (4971S) antibodies were purchased from Cell Signaling Technology and human anti-actin (622102) antibody was purchased from BioLegend.

Techniques: Transfection, Plasmid Preparation, Lysis, Western Blot, Electroporation, Expressing

Journal: Life Science Alliance

Article Title: Human immunodeficiency virus type 1 impairs sumoylation

doi: 10.26508/lsa.202101103

Figure Lengend Snippet: (A) Western blots show SUMO conjugate levels in total leukocytes of a representative ART-naive HIV-1–infected individual (HIV(+)), in comparison with an uninfected individual (HIV(−)). Western blots were performed as described in and in the Materials and Methods section; sumoylated proteins were detected using human anti-SUMO1 and anti-SUMO2/3 antibodies. Actin: loading control (important note: equal amounts [20 μg] of protein were loaded in each well to allow fair comparison of SUMO profiles between HIV(−) and HIV(+) samples). Graphs (right panel) show densitometric quantifications of SUMO signals normalized to actin expression. Data are presented as mean ± SEM (n = 15 individuals for SUMO1, n = 19 individuals for SUMO2/3), P -values are indicated (using t test assuming unequal variances). Asterisks denote statistical significance. (B) The contribution of CD4 + cells to total leukocyte sumoylation was assessed by Western blot after ex vivo depletion of the former from the peripheral blood (leukocytes) of HIV-negative individuals. Total leukocytes (total: before depletion) and the CD4 + cell-deprived fraction (CD4(−)) are shown for comparison for a representative individual. Graphs show densitometric quantifications of SUMO signals normalized to actin expression. Data are presented as mean ± SEM (n = 4 individuals), P -values were calculated using t test assuming unequal variances, ns, not significant. Sumoylation in the CD4 + fraction is shown in . (C) Western blot shows leukocyte sumoylation profiles of a representative HIV-1–infected individual before (labeled as naive) and after anti-retroviral therapy (labeled as ART), with respect to that of an uninfected control individual (HIV(−)). Patient received ART for 3 mo after which the HIV-1 viral load became undetectable, yet the CD4 + count did not considerably rise. Graph shows densitometric quantifications of SUMO signals normalized to actin expression. Data are presented as mean ± SEM (n = 3 individuals), P -values are indicated (using t test assuming unequal variances). Asterisks denote statistical significance. (D) Western blot analysis of UBA2 in total leukocytes of a representative ART-naive HIV-1–infected individual (HIV(+)), in comparison with an uninfected individual (HIV(−)). 3 HIV(+) and 3 HIV(−) individuals were analyzed; graph shows densitometric quantifications of UBA2 signals normalized to actin expression (data are presented as mean ± SEM, n = 3 individuals, asterisks denote statistical significance; P -value is indicated, using t test assuming unequal variances).

Article Snippet: Human anti-SUMO1 (4930S) and anti-SUMO2/3 (4971S) antibodies were purchased from Cell Signaling Technology and human anti-actin (622102) antibody was purchased from BioLegend.

Techniques: Western Blot, Infection, Expressing, Ex Vivo, Labeling