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human anti sumo1 4930s (Cell Signaling Technology Inc)

Name: human anti sumo1 4930s
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Cell Signaling Technology Inc human anti sumo1 4930s

(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 <t>SUMO1</t> 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.

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Article Title: Human immunodeficiency virus type 1 impairs sumoylation

Journal: Life Science Alliance

doi: 10.26508/lsa.202101103

Figure Legend 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.

Techniques Used: Expressing, Western Blot, Transfection, Plasmid Preparation

(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.

Figure Legend 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.

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

(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 .

Figure Legend 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 .

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

(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.

Figure Legend 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.

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

$(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).$
Figure Legend 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).

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

Image Search Results

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

$(A) Immunoprecipitation of Cas9 expressed in HEK293 cells reveals Cas9 conjugation by SUMO1 and SUMO2/3 peptides. HEK293 cells were transfected with doxycycline (Dox)-inducible FLAG-tagged Cas9 (from Streptococcus pyogenes ) along with GFP-tagged SUMO paralogs. Pull-down of FLAG-–Cas9 was followed by Western blot analysis with SUMO1, SUMO2/3, or GFP antibodies, which yielded a smear pattern corresponding to sumoylated Cas9 (Cas9–SUMO, highlighted by brackets). Negative controls are shown in (pull-down with a nonspecific IgG, also pull-down from cells expressing GFP-SUMO only) and in . (B) Verification of Cas9 sumoylation by His pull-down. HEK293 cells were transfected with FLAG–Cas9 along with histidine-tagged SUMO paralogs (His-SUMO1 or His–SUMO2/3). Nickel-purified His-SUMO conjugates were probed with a FLAG antibody to detect sumoylated Cas9 forms (highlighted by asterisks; a small fraction of unmodified Cas9 adsorbs non-specifically to beads [arrowhead]). (C) Immunoprecipitation of Cas9 expressed in HEK293 cells reveals Cas9 conjugation by endogenous SUMO1 and SUMO2/3. HEK293 cells were transfected with doxycycline (Dox)-inducible FLAG-tagged Cas9 only. (D) Proximity ligation (Duolink) assays probe Cas9 modification by endogenous SUMO1 and SUMO2/3 peptides, as well as physical interaction with the UBC9 E2 SUMO conjugating enzyme in HEK293 cells stably expressing Streptococcus pyogenes Cas9. Z-stack projections are shown. Nuclei were stained with DAPI. Quantifications of positive Duolink signals are shown in the graph. n > 25 cells per experiment. Data represent mean value from two experiments per condition, including the negative controls using a single antibody of a given Duolink pair, ± SEM (**** P < 0.0001, unpaired t test). Representative confocal images of the negative controls are shown in . For simplicity, P -values between the following groups were not included in the graph: P Cas9-SUMO1 & Cas9 < 0.0001, P Cas9-SUMO1 & SUMO1 < 0.0001, P Cas9-SUMO2/3 & Cas9 < 0.0001, P Cas9-SUMO2/3 & SUMO2/3 < 0.0001, P Cas9-Ubc9 & Cas9 < 0.0001, P Cas9-Ubc9 & Ubc9 < 0.0001.$

Journal: Life Science Alliance

Article Title: Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding

doi: 10.26508/lsa.202101078

Figure Lengend Snippet: (A) Immunoprecipitation of Cas9 expressed in HEK293 cells reveals Cas9 conjugation by SUMO1 and SUMO2/3 peptides. HEK293 cells were transfected with doxycycline (Dox)-inducible FLAG-tagged Cas9 (from Streptococcus pyogenes ) along with GFP-tagged SUMO paralogs. Pull-down of FLAG-–Cas9 was followed by Western blot analysis with SUMO1, SUMO2/3, or GFP antibodies, which yielded a smear pattern corresponding to sumoylated Cas9 (Cas9–SUMO, highlighted by brackets). Negative controls are shown in (pull-down with a nonspecific IgG, also pull-down from cells expressing GFP-SUMO only) and in . (B) Verification of Cas9 sumoylation by His pull-down. HEK293 cells were transfected with FLAG–Cas9 along with histidine-tagged SUMO paralogs (His-SUMO1 or His–SUMO2/3). Nickel-purified His-SUMO conjugates were probed with a FLAG antibody to detect sumoylated Cas9 forms (highlighted by asterisks; a small fraction of unmodified Cas9 adsorbs non-specifically to beads [arrowhead]). (C) Immunoprecipitation of Cas9 expressed in HEK293 cells reveals Cas9 conjugation by endogenous SUMO1 and SUMO2/3. HEK293 cells were transfected with doxycycline (Dox)-inducible FLAG-tagged Cas9 only. (D) Proximity ligation (Duolink) assays probe Cas9 modification by endogenous SUMO1 and SUMO2/3 peptides, as well as physical interaction with the UBC9 E2 SUMO conjugating enzyme in HEK293 cells stably expressing Streptococcus pyogenes Cas9. Z-stack projections are shown. Nuclei were stained with DAPI. Quantifications of positive Duolink signals are shown in the graph. n > 25 cells per experiment. Data represent mean value from two experiments per condition, including the negative controls using a single antibody of a given Duolink pair, ± SEM (**** P < 0.0001, unpaired t test). Representative confocal images of the negative controls are shown in . For simplicity, P -values between the following groups were not included in the graph: P Cas9-SUMO1 & Cas9 < 0.0001, P Cas9-SUMO1 & SUMO1 < 0.0001, P Cas9-SUMO2/3 & Cas9 < 0.0001, P Cas9-SUMO2/3 & SUMO2/3 < 0.0001, P Cas9-Ubc9 & Cas9 < 0.0001, P Cas9-Ubc9 & Ubc9 < 0.0001.

Article Snippet: The following antibodies were used for immunoprecipitations, immunoblots, PLA, and mass spectrometry: human anti-SUMO1 (#4930; CST), human anti-SUMO2/3 (ab3742; Abcam), anti-GFP (sc-9996; Santa Cruz), human anti-ubiquitin (Clone FK2; R&D Systems), anti-FLAG (#F1804; Sigma-Aldrich), human anti-UBC9 (#4786; CST), anti-Cas9, (#7A9; BioLegend), human anti-PSMA5 (#2457; CST), human anti-actin (#622102; BioLegend), and human anti-GAPDH (sc-32233; Santa Cruz).

Techniques: Immunoprecipitation, Conjugation Assay, Transfection, Western Blot, Expressing, Purification, Ligation, Modification, Stable Transfection, Staining

(A) Assessment of Cas9 sumoylation (bracket) by immunoprecipitation, as in . HEK293 cells were transfected with the indicated constructs. This experiment includes two extra negative controls: cells transfected with GFP-SUMO1 alone or with GFP-SUMO2/3 alone. (B) Proximity ligation (Duolink) assays in HK-2 cells verify Cas9 modification by endogenous SUMO1 and SUMO2/3 peptides, as well as physical interaction with the UBC9 E2 SUMO conjugating enzyme. Cas9 was stably expressed in HK-2 cells. Z-stack projections are shown. Nuclei were stained with DAPI. Quantifications of positive Duolink signals are shown in the graph. n = 30 cells per experiment. Data represent mean value from two experiments per condition, including the negative controls employing a single antibody of a given Duolink pair, ± SEM (**** P < 0.0001, unpaired t test). For simplicity, P -values between the following groups were not included in the graph: P Cas9-SUMO1 & Cas9 < 0.0001, P Cas9-SUMO1 & SUMO1 < 0.0001, P Cas9-SUMO2/3 & Cas9 < 0.0001, P Cas9-SUMO2/3 & SUMO2/3 < 0.0001, P Cas9-Ubc9 & Cas9 < 0.0001, P Cas9-Ubc9 & Ubc9 < 0.0001. (C) Representative confocal images of the negative controls for PLA experiments shown in and (B). Nuclei were stained with DAPI.

Journal: Life Science Alliance

Article Title: Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding

doi: 10.26508/lsa.202101078

Figure Lengend Snippet: (A) Assessment of Cas9 sumoylation (bracket) by immunoprecipitation, as in . HEK293 cells were transfected with the indicated constructs. This experiment includes two extra negative controls: cells transfected with GFP-SUMO1 alone or with GFP-SUMO2/3 alone. (B) Proximity ligation (Duolink) assays in HK-2 cells verify Cas9 modification by endogenous SUMO1 and SUMO2/3 peptides, as well as physical interaction with the UBC9 E2 SUMO conjugating enzyme. Cas9 was stably expressed in HK-2 cells. Z-stack projections are shown. Nuclei were stained with DAPI. Quantifications of positive Duolink signals are shown in the graph. n = 30 cells per experiment. Data represent mean value from two experiments per condition, including the negative controls employing a single antibody of a given Duolink pair, ± SEM (**** P < 0.0001, unpaired t test). For simplicity, P -values between the following groups were not included in the graph: P Cas9-SUMO1 & Cas9 < 0.0001, P Cas9-SUMO1 & SUMO1 < 0.0001, P Cas9-SUMO2/3 & Cas9 < 0.0001, P Cas9-SUMO2/3 & SUMO2/3 < 0.0001, P Cas9-Ubc9 & Cas9 < 0.0001, P Cas9-Ubc9 & Ubc9 < 0.0001. (C) Representative confocal images of the negative controls for PLA experiments shown in and (B). Nuclei were stained with DAPI.

Techniques: Immunoprecipitation, Transfection, Construct, Ligation, Modification, Stable Transfection, Staining

(A, B) Conjugation status by SUMO2/3 (A) or by SUMO1 (B) of the 10 different FLAG–Cas9 constructs, each carrying a specific lysine-to-arginine mutation that renders one of the consensus motifs defective, as assessed by immunoprecipitation. HEK293 cells were transfected with the indicated constructs. WT: wild-type Cas9. (C) Conjugation of Cas9–K848R by the endogenously expressed SUMO1 peptide was assessed by immunoprecipitation. HEK293 cells were transfected with the indicated constructs. (D) Conjugation of Cas9–K848R by endogenous SUMO1 was assessed by PLA in HEK293 cells stably expressing wild-type Cas9 (WT) or its K848R mutant. Nuclei were stained with DAPI. Quantifications of positive Duolink signals are shown in the graph. n > 30 cells per experiment. Data represent mean value from two experiments per condition, including the negative controls using a single antibody of a given Duolink pair, ± SEM (* P < 0.05, unpaired t test). Results point to a slight yet negligible decrease in SUMO1 conjugation with the K848R mutant. For simplicity, P -values between the following groups were not included in the graph: P Cas9WT-SUMO1 & Cas9 < 0.0001, P Cas9WT-SUMO1 & SUMO1 < 0.0001, P Cas9K848R-SUMO1 & Cas9 < 0.0001, P Cas9K848R-SUMO1 & SUMO1 < 0.0001.

Journal: Life Science Alliance

Article Title: Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding

doi: 10.26508/lsa.202101078

Figure Lengend Snippet: (A, B) Conjugation status by SUMO2/3 (A) or by SUMO1 (B) of the 10 different FLAG–Cas9 constructs, each carrying a specific lysine-to-arginine mutation that renders one of the consensus motifs defective, as assessed by immunoprecipitation. HEK293 cells were transfected with the indicated constructs. WT: wild-type Cas9. (C) Conjugation of Cas9–K848R by the endogenously expressed SUMO1 peptide was assessed by immunoprecipitation. HEK293 cells were transfected with the indicated constructs. (D) Conjugation of Cas9–K848R by endogenous SUMO1 was assessed by PLA in HEK293 cells stably expressing wild-type Cas9 (WT) or its K848R mutant. Nuclei were stained with DAPI. Quantifications of positive Duolink signals are shown in the graph. n > 30 cells per experiment. Data represent mean value from two experiments per condition, including the negative controls using a single antibody of a given Duolink pair, ± SEM (* P < 0.05, unpaired t test). Results point to a slight yet negligible decrease in SUMO1 conjugation with the K848R mutant. For simplicity, P -values between the following groups were not included in the graph: P Cas9WT-SUMO1 & Cas9 < 0.0001, P Cas9WT-SUMO1 & SUMO1 < 0.0001, P Cas9K848R-SUMO1 & Cas9 < 0.0001, P Cas9K848R-SUMO1 & SUMO1 < 0.0001.

Techniques: Conjugation Assay, Construct, Mutagenesis, Immunoprecipitation, Transfection, Stable Transfection, Expressing, Staining

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