Journal: Frontiers in immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: FIGURE 2 The effect of acute ethanol-exposure on mouse bone marrow derived macrophages (BMDM). Phagocytosis in BMDM exposed to vehicle or ethanol ± LPS to study phagocytosis using pHrodo bioparticles and SIRT2 expression. (A) Representative images of intracellular pHrodo bioparticles in vehicle or Ethanol-exposed WT-BMDM ± LPS. (B) Fluorescence quantification of pHrodo bioparticles in WT-BMDM (n=4 repetitions/group; * p<0.05). (C) SIRT2 protein expression detected by western blot in Vehicle- or Ethanol-exposed BMDM cells ± LPS. (D) Western blot image quantification of SIRT2 protein blot in vehicle or ethanol-exposed BMDM cells ± LPS (n = 4 blots/group; * p < 0.05). (E) Representative images of pHrodo bioparticles in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. (F) Fluorescence quantification of pHrodo bioparticles in Ethanol-exposed WT-BMDM, and SIRT2KO-BMDM ± LPS. * p < 0.05. (G) Representative images of pHrodo bioparticles in Ethanol-exposed WT-BMDM, co-treated with SIRT2 inhibitor AK-7 or DMSO ± LPS. (H) Fluorescence quantification of pHrodo bioparticles in Ethanol-exposed WT-BMDM with AK-7/DMSO ± LPS stimulation. * p < 0.05.

Article Snippet: Antibodies and reagents used for western blot and immunocytochemistry PFKP (Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Antirabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PUN), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Derivative Assay, Expressing, Fluorescence, Western Blot

Journal: Frontiers in immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: FIGURE 4 The effect of acute ethanol-exposure-induced SIRT2 on PFKP expression in macrophages. (A) PFKP expression in WT-BMDM exposed to vehicle or ethanol ± LPS by western blot analysis. (B) PFKP western blot image quantification of PFKP protein in vehicle vs. ethanol exposed WT-BMDM, Y axis represents fold of vehicle-LPS (fold of vehicle control) (n = 4 blots; * p < 0.05). (C) PFKP expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. (D) Western blot image quantification of PFKP protein in ethanol exposed WT-BMDM and SIRT2KO-BMDM. Y axis represents fold of Ethanol-exposed WT-LPS (fold of WT- ethanol control) (n = 4 blots; * p < 0.05). (E) PFKP expression in Ethanol-exposed WT- BMDM treated with AK-7 or DMSO ± LPS. (F) Western blot image quantification of PFKP in AK-7 vs. DMSO treated-Ethanol-exposed WT-BMDM ± LPS, Y axis represents fold of Ethanol-exposed WT-LPS (fold of WT- ethanol control) (n = 4 blots; * p < 0.05).

Article Snippet: Antibodies and reagents used for western blot and immunocytochemistry PFKP (Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Antirabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PUN), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: FIGURE 5 SIRT2-PFKP in vivo and in vitro interaction. (A) RAW264.7 cell macrophages (RAW) ± LPS. IP of whole-cell lysates using an anti-SIRT2 antibody followed by IB analysis of PFKP and SIRT2. IP with isotype IgG control antibody was used as a negative control. (B) Western blot analysis of PFKP and SIRT2 in the whole cell lysate used as input for the SIRT2 IP. (C) In-vitro interaction between SIRT2 and PFKP using SPR. SIRT2 protein immobilized onto sensor chip and the PFKP was flowed at various concentration (15.62, 31.25, 62.5, 125, 250, 500 and 1000nM). The response units on Y axis (RU) represent quantitative assessment of protein-protein interaction. (D) wtPFKP and control plasmid transfection and IP, using turbo-GFP-trap in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of acetyl lysine, turbo-GFP-wtPFKP and control for PFKP (turbo-GFP). HEK293T cell lysate without transfection used as a negative control. (E) Western blot analysis of control-PFKP (Turbo-GFP), wtPFKP (Turbo-GFP-wtPFKP), DDK-SIRT2 and CPA in whole cell lysate used as input for the turbo-GFP IP. (F) wtPFKP transfection and IP using magnetic-TUBEs in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of ubiquitination. (G) Western blot analysis of turbo- GFP wtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate used as an input for the TUBE IP.

Article Snippet: Antibodies and reagents used for western blot and immunocytochemistry PFKP (Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Antirabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PUN), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: In Vivo, In Vitro, Control, Negative Control, Western Blot, Concentration Assay, Plasmid Preparation, Transfection, Ubiquitin Proteomics

Journal: Frontiers in immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: FIGURE 6 Effect of K394R mutation on PFKP. (A, B) HEK293T cells transfected with wtPFKP/mtPFKP in the presence or absence of SIRT2. Western blot analysis of Turbo-GFP-wtPFKP, DDK-SIRT2, turbo-GFP (control for wtPFKP plasmid transfected) and CPA. (C) mtPFKP transfection and IP using turbo-GFP-trap in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of acetyl lysine, turbo-GFP-mtPFKP and turbo-GFP (control for wtPFKP plasmid transfected). Pulldown with HEK293T cell lysate without transfection was used as a negative control. (D) Western blot analysis of turbo-GFP mtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate, used as an input for the turbo-GFP IP. (E) mtPFKP transfection and IP using magnetic-TUBEs in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of ubiquitination. (F) Western blot analysis of turbo-GFP mtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate used as input for the TUBE IP.

Article Snippet: Antibodies and reagents used for western blot and immunocytochemistry PFKP (Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Antirabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PUN), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Mutagenesis, Transfection, Western Blot, Control, Plasmid Preparation, Negative Control, Ubiquitin Proteomics

Journal: Frontiers in immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: FIGURE 11 The effect of acute ethanol-exposure on SIRT2 expression and the effect of AK-7 on LC3-associated phagocytosis in Ethanol-exposed Human macrophages. Human macrophages were exposed to vehicle or ethanol ± LPS to study SIRT2 expression and LAP. SIRT2 expression was analyzed by immunostaining (A) Representative images of SIRT2 immunostaining in vehicle or Ethanol-exposed human macrophages ± LPS. (B) Fluorescence quantification of SIRT2 immunostaining in human macrophages (n=4; *p<0.05). (C) LAP in human macrophages with vehicle or ethanol-exposure ± LPS. Representative images of intracellular pHrodo bioparticles during phagocytosis and co-stained for LC3. (D) For each image, the co-localization of intracellular pHrodo (red) and LC3 (green) were determined as LAP and divided by the total numbers of nuclei. Graph represents fluorescence quantification of LAP in human macrophages (n=4; * p<0.05). (E) Ethanol-exposed human macrophages were co-treated with SIRT2 inhibitor AK-7 or DMSO ± LPS. Representative images of intracellular pHrodo bioparticles during phagocytosis and stained for LC3 to study LAP. (F) Graph represents fluorescence quantification of LAP in Ethanol-exposed human macrophages ± AK-7 ± LPS stimulation (n=4; * p<0.05).

Article Snippet: Antibodies and reagents used for western blot and immunocytochemistry PFKP (Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Antirabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PUN), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Expressing, Immunostaining, Fluorescence, Staining

Journal: Cell reports

Article Title: SIRT2 promotes BRCA1-BARD1 heterodimerization through deacetylation

doi: 10.1016/j.celrep.2021.108921

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-SIRT2 , ThermoFisher custom made; This paper , N/A.

Techniques: Recombinant, Software

Journal: eLife

Article Title: tRNA synthetase counteracts c-Myc to develop functional vasculature

doi: 10.7554/eLife.02349

Figure Lengend Snippet: ( A ) SerRS specifically interacts with SIRT2 but not SIRT1. HEK 293 cells were co-transfected with plasmids expressing Flag-tagged SerRS and V5-tagged SIRT1 or SIRT2. Cell lysate was immunoprecipitated with anti-V5 (top panel), anti-Flag (bottom panel) antibodies or control IgG. The experiment was followed by Western blot analysis to detect the interaction between SerRS and SIRT1/SIRT2 using anti-Flag and anti-V5 antibodies. ( B ) GST-pull down assay to show that SerRS/SIRT2 interaction is direct and that the interaction is mediated by the catalytic domain of SerRS. Full-length SerRS or its domain fragments were fused with GST at N-termini to pull down purified His-tagged SIRT2. SIRT2 was detected by Western blot analysis using anti-His 6 antibody, and the GST fusion proteins attached on the Glutathione-Sepharose beads were analyzed using ponceau S staining. TBD: tRNA-binding domain; CD: catalytic domain; UNE-S: C-terminal appended domain. ( C ) Mapping study to identify the SerRS binding sites on SIRT2. V5-tagged full-length SIRT2 or its truncated fragments was co-transfected with Flag-tagged SerRS into HEK 293 cells. SIRT2 proteins were immunoprecipitated with anti-V5 antibody and the SIRT2-bound SerRS proteins were detected by Western blot using anti-Flag antibody. ( D ) Illustration of the SerRS binding sites on the structure of SIRT2. Two SerRS binding sites (Gly52-Asp60, Trp337-Ser356) are highlighted in red. The catalytic domain of SIRT2 is in green, while the partially disordered N- and C-terminal regions are in yellow and purple, respectively. The gray dash line represents a disordered internal region. ( E ) Effect of SerRS on SIRT2 deacetylation activity. Recombinant human SIRT2 (1 µM) were incubated with purified SerRS (concentration measured as monomer) at the indicated ratios. The deacetylase activities of SIRT2 were measured by using a substrate peptide with one end coupled to a fluorophore and the other end to a quencher. An internal acetylated lysine residue serves as the substrate of SIRT2, and the deacetylation allows the peptide to be cleaved by a lysylendopeptidase to release the fluorophore from the quencher to emit fluorescence. Therefore, the SIRT2 acitivity was measured by monitoring the fluororescence intensity (excitation at 490 nm and emission at 530 nm). A reaction without NAD + (NAD + [−]) was performed as a negative control. DOI: http://dx.doi.org/10.7554/eLife.02349.016

Article Snippet: Anti-c-Myc, anti-SIRT2, anti-SIRT1, and anti-α-tubulin antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Purification, Staining, Binding Assay, Activity Assay, Recombinant, Incubation, Concentration Assay, Histone Deacetylase Assay, Fluorescence, Negative Control

Journal: eLife

Article Title: tRNA synthetase counteracts c-Myc to develop functional vasculature

doi: 10.7554/eLife.02349

Figure Lengend Snippet: Approximately 10 7 HEK 293 cells were lysed and the supernatant were immunoprecipicated with mouse monoclonal anti-SerRS antibody or the control mouse IgG. The co-immunoprecipitated proteins were analyzed by Western blot analysis using anti-SerRS and anti-SIRT2 antibodies, respectively. DOI: http://dx.doi.org/10.7554/eLife.02349.017

Article Snippet: Anti-c-Myc, anti-SIRT2, anti-SIRT1, and anti-α-tubulin antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: eLife

Article Title: tRNA synthetase counteracts c-Myc to develop functional vasculature

doi: 10.7554/eLife.02349

Figure Lengend Snippet: ChIP assay to detect the impact of SerRS and GlyRS (as a control) on SIRT2 binding to the VEGFA promoter. HEK 293 cells were transfected with plasmids expressing the indicated shRNA for knocking down the expression of SerRS or GlyRS and subjected to ChIP analysis 48 hr post-transfection. The normalized results are represented as fold change of immunoprecipitated DNA by anti-SerRS vs anti-SIRT2 and are shown as means ± SEM (n = 3). DOI: http://dx.doi.org/10.7554/eLife.02349.018

Article Snippet: Anti-c-Myc, anti-SIRT2, anti-SIRT1, and anti-α-tubulin antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Binding Assay, Transfection, Expressing, shRNA, Immunoprecipitation

Journal: eLife

Article Title: tRNA synthetase counteracts c-Myc to develop functional vasculature

doi: 10.7554/eLife.02349

Figure Lengend Snippet: ( A ) ChIP assay to show that overexpression of SerRS reduces histone H4 acetylation level on the VEGFA promoter. HEK 293 cells were transfected with plasmids expressing SerRS, GlyRS or empty vector. The cell lysates were subjected to local ChIP analysis using anti-H4K16Ac (acetylated H4 at K16), anti-H4 (total), or anti-SerRS antibodies and a primer set targeting the VEGFA promoter. The amounts of DNA immunoprecipitated by anti-H4K16Ac antibody were normalized to those by anti-H4 antibody prior to fold change calculation. Inset: the normalized amounts of DNA immunoprecipitated by anti-SerRS. All data were shown as means ± SEM (n = 3). ( B ) ChIP assay to show that knock down of SerRS expression or exclusion of SerRS from the nucleus increases histone H4 acetylation level on the VEGFA promoter. HEK 293 cells were transfected with plasmids expressing the indicated molecules and subjected to local ChIP analysis as described above. As a control, GlyRS expression was knocked down but had no effect on H4 acetylation. ( C ) Effect of SIRT2 expression on the transcriptional repressor activity of SerRS as measured by VEGFA expression. HEK 293 cells were co-transfected with plasmids expressing shRNAs targeting SIRT1, SIRT2 or control shRNA and plasmids expressing SerRS, GlyRS or empty vector for 36 hr. The VEGFA expression levels were determined by using real-time RT-qPCR and are shown as means ± SEM (n = 3). ( D ) Effect of SIRT2-specific inhibitor on the transcriptional repressor activity of SerRS as measured by VEGFA expression. HEK 293 cells were transfected with plasmids expressing SerRS, GlyRS or empty vector. SIRT2-specific inhibitor AGK2 (10 µM, final concentration) or SIRT1-specific inhibitor EX-527 (1 µm, final concentration) or solvent alone (DMSO) was added to the cell culture media 2 hr post-transfection. VEGFA expression levels were measured 24 hr post-transfection by using real-time RT-qPCR and are shown as means ± SEM (n = 3). ( E ) Functional correlation between SerRS and SIRT2 in zebrafish. The percentage of Tg(Fli1a:EGFP) zebrafish embryos showing different ISV phenotypes at 3 days post fertilization after the injection of morpholinos targeting SerRS (SerRS-MO), Sirt1 (Sirt1-MO), Sirt2 (Sirt2-MO), or a control morpholino (Control-MO) are illustrated. Scale bars represent 0.25 mm. *p<0.0001 vs Control-MO, **p>0.1 vs Control-MO. ( F ) The effects of knocking down SerRS, Sirt2, or Sirt1 in zebrafish on Vegfa expression were examined by real-time RT-qPCR at 1 day post fertilization after injection of morpholinos as indicated. Data are shown as means ± SEM (n = 10–15). DOI: http://dx.doi.org/10.7554/eLife.02349.019

Article Snippet: Anti-c-Myc, anti-SIRT2, anti-SIRT1, and anti-α-tubulin antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Activity Assay, shRNA, Quantitative RT-PCR, Concentration Assay, Cell Culture, Functional Assay, Injection

Journal: eLife

Article Title: tRNA synthetase counteracts c-Myc to develop functional vasculature

doi: 10.7554/eLife.02349

Figure Lengend Snippet: ( A ) Western blot analysis to confirm the efficiency of shRNAs in knocking down endogenous GlyRS (sh-GlyRS) or SerRS (sh-SerRS) expression in HEK 293 cells. ( B ) Western blot analysis to confirm the efficiency of shRNAs in knocking down endogenous SIRT1 (sh-SIRT1) or SIRT2 (sh-SIRT2) expression in HEK 293 cells. DOI: http://dx.doi.org/10.7554/eLife.02349.020

Article Snippet: Anti-c-Myc, anti-SIRT2, anti-SIRT1, and anti-α-tubulin antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Expressing

Journal: eLife

Article Title: tRNA synthetase counteracts c-Myc to develop functional vasculature

doi: 10.7554/eLife.02349

Figure Lengend Snippet: ( A ) The design of Sirt2-MO and the expected mRNA/protein products. The Sirt2-MO targets the exon4-intron4 splicing site of Sirt2 precursor mRNA to potentially generate two alternatively spliced mRNA products (SB) that would be translated into non-functional proteins. ( B ) RT-PCR analysis confirming the dramatic decrease of WT Sirt2 mature mRNA and the expected alternative splicing variants (SB) as the result of injecting Sirt2-MO. DOI: http://dx.doi.org/10.7554/eLife.02349.021

Article Snippet: Anti-c-Myc, anti-SIRT2, anti-SIRT1, and anti-α-tubulin antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Functional Assay, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: tRNA synthetase counteracts c-Myc to develop functional vasculature

doi: 10.7554/eLife.02349

Figure Lengend Snippet: Nuclear SerRS binds to the VEGFA promoter at the identified SerRS binding site (SBS) and recruits the SIRT2 histone deacetylase to condense the local chromatin to shut down VEGFA expression. These tandem actions of SerRS symmetrically offset the VEGFA-promoting actions of c-Myc to maintain a delicate balance for the development of a functional vasculature. DOI: http://dx.doi.org/10.7554/eLife.02349.022

Article Snippet: Anti-c-Myc, anti-SIRT2, anti-SIRT1, and anti-α-tubulin antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Binding Assay, Histone Deacetylase Assay, Expressing, Functional Assay

Journal: Aging (Albany NY)

Article Title: Melatonin ameliorates the advanced maternal age-associated meiotic defects in oocytes through the SIRT2-dependent H4K16 deacetylation pathway

doi: 10.18632/aging.102703

Figure Lengend Snippet: Effects of melatonin administration on SIRT2 expression in oocytes from old mice. ( A ) Western blot analysis shows the SIRT2 expression in GV oocytes from young, old and old+Mel mice. Actin served as a loading control. Band intensity was calculated using ImageJ software. ( B ) Representative confocal images of young, old and old+Mel MII oocytes stained with SIRT2 antibody (green) and counterstained with propidium iodide (red) for chromosomes. ( C ) Quantification of the relative fluorescence intensity of SIRT2 in oocytes in ( B ). Each data point represents an oocyte (n=10 for each group). *P<0.05 vs. controls. Scale bar: 50 μm.

Article Snippet: Antibodies were purchased from the following sources: mouse monoclonal anti-α-tubulin-FITC antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: T6074); rabbit polyclonal anti-SIRT2 antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: S8447); mouse monoclonal anti-β-actin antibodies (St. Louis, MO, USA; Cat#: A5441) were purchased from Sigma; rabbit monoclonal anti-H4K16ac antibodies were purchased from Abcam (Cambridge, MA, USA; Cat#: ab109463); mouse monoclonal anti-Myc antibodies were purchased from Abcam (Cambridge, MA, USA; Cat#: ab18185).

Techniques: Expressing, Western Blot, Software, Staining, Fluorescence

Journal: Aging (Albany NY)

Article Title: Melatonin ameliorates the advanced maternal age-associated meiotic defects in oocytes through the SIRT2-dependent H4K16 deacetylation pathway

doi: 10.18632/aging.102703

Figure Lengend Snippet: SIRT2 knockdown abolishes the effects of melatonin on meiotic phenotypes in aged oocytes. ( A ) Schematic illustration of the experimental protocol to check whether SIRT2 mediates the effects of melatonin on aged oocyte. ( B ) The efficiency of SIRT2 knockdown was verified by Western blot. Actin served as a loading control. Band intensity was calculated using ImageJ software. ( C ) Representative images of spindle/chromosome organization in young, old, old+Mel and old+Mel+siSirt2 oocytes are shown. Arrows indicate the disorganized spindle and arrowheads indicate the misaligned chromosomes. ( D ) Quantification of young, old, old+Mel and old+Mel+siSirt2 oocytes with abnormal spindle/chromosomes. Error bars indicate ± SD. *P<0.05 vs. controls. Scale bars: 50 μm.

Article Snippet: Antibodies were purchased from the following sources: mouse monoclonal anti-α-tubulin-FITC antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: T6074); rabbit polyclonal anti-SIRT2 antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: S8447); mouse monoclonal anti-β-actin antibodies (St. Louis, MO, USA; Cat#: A5441) were purchased from Sigma; rabbit monoclonal anti-H4K16ac antibodies were purchased from Abcam (Cambridge, MA, USA; Cat#: ab109463); mouse monoclonal anti-Myc antibodies were purchased from Abcam (Cambridge, MA, USA; Cat#: ab18185).

Techniques: Western Blot, Software

Journal: Aging (Albany NY)

Article Title: Melatonin ameliorates the advanced maternal age-associated meiotic defects in oocytes through the SIRT2-dependent H4K16 deacetylation pathway

doi: 10.18632/aging.102703

Figure Lengend Snippet: H4K16 hyperacetylation in old and SIRT2-depleted oocytes. ( A ) Control and SIRT2 knockdown (siSirt2) oocytes were immunolabeled with H4K16ac antibody (green) and counterstained with propidium iodide (red) for DNA. Representative confocal sections are shown. ( B ) Quantification of fluorescence intensity of acetylated H4K16 in control and siSirt2 oocytes. (n=10 for each group). ( C ) Young, old and old+Mel oocytes were immunolabeled with H4K16ac antibody (green) and counterstained with propidium iodide (red) for DNA. Representative confocal sections are shown. ( D ) Quantification of fluorescence intensity of acetylated H4K16 in young, old and old+Mel oocytes (n=10 for each group). Data are expressed as the mean ± SD from three independent experiments. *P<0.05 vs. controls. Scale bars: 5 μm.

Article Snippet: Antibodies were purchased from the following sources: mouse monoclonal anti-α-tubulin-FITC antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: T6074); rabbit polyclonal anti-SIRT2 antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: S8447); mouse monoclonal anti-β-actin antibodies (St. Louis, MO, USA; Cat#: A5441) were purchased from Sigma; rabbit monoclonal anti-H4K16ac antibodies were purchased from Abcam (Cambridge, MA, USA; Cat#: ab109463); mouse monoclonal anti-Myc antibodies were purchased from Abcam (Cambridge, MA, USA; Cat#: ab18185).

Techniques: Immunolabeling, Fluorescence

Journal: Aging (Albany NY)

Article Title: Melatonin ameliorates the advanced maternal age-associated meiotic defects in oocytes through the SIRT2-dependent H4K16 deacetylation pathway

doi: 10.18632/aging.102703

Figure Lengend Snippet: Diagram illustrating the proposed mechanisms mediating the beneficial effects of melatonin on meiotic phenotypes of aged oocytes. Melatonin supplement induces SIRT2 expression in oocytes from old mice, which in turn decreases the acetylation levels of H4K16, and thereupon promoting the establishment of proper kinetochore-microtubule attachment and the assembly of meiotic apparatus.

Article Snippet: Antibodies were purchased from the following sources: mouse monoclonal anti-α-tubulin-FITC antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: T6074); rabbit polyclonal anti-SIRT2 antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: S8447); mouse monoclonal anti-β-actin antibodies (St. Louis, MO, USA; Cat#: A5441) were purchased from Sigma; rabbit monoclonal anti-H4K16ac antibodies were purchased from Abcam (Cambridge, MA, USA; Cat#: ab109463); mouse monoclonal anti-Myc antibodies were purchased from Abcam (Cambridge, MA, USA; Cat#: ab18185).

Techniques: Expressing