Journal: Viruses

Article Title: NAD+ Suppresses EV-D68 Infection by Enhancing Anti-Viral Effect of SIRT1

doi: 10.3390/v17020175

Figure Lengend Snippet: Viral infection regulated the expression of enzymes in the NAD+ salvage pathway, leading to a reduction in NAD+ level. ( a ) RD cells were cultured in 6-well plates and infected with EV-D68 Fermon at an MOI of 0 or 1 for 24 h. WST—detection of NAD+ in cells by WST-8 reaction colorimetry. ( b – e ) RD and A549 cells were infected with EV-D68 Fermon at an MOI of 1. Then, 18 h later, the expression changes of NAD+ synthesis genes (Nmnat1 to 3, Nampt, and Nmrk2) and NAD+ consuming genes (Sirt1 to 7, Parp1 to 6, Parp9, 10, 12, 14, and CD38) were measured using RT-qPCR. ( f ) A549 cells were infected with EV-D68 Fermon at an MOI of 1. Then, 18 h later, the protein levels of VP1, SIRT1, SIRT2, NAMPT, NMNAT2, and CD38, compared to the control β-actin, were determined using Western blot. Data in all quantitative panels are normalized based on β-actin or GAPDH presented as the mean ± SD of n = 3 replicates. Error bars indicate SD (n = 3). “−” represents absence, and “+” represents presence. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The primary antibodies used were mouse anti-EV-D68 VP1 (1:2000, GeneTex, San Antonio, TX, USA), rabbit anti-NAMPT (1:500, Sangon, China), rabbit anti-SIRT1 (1:2000, GeneTex, San Antonio, TX, USA), rabbit anti-NMANT2 (1:2000, Sangon, China), rabbit anti-CD38 (1:2000, Sangon, China), rabbit anti-SIRT2 (1:2000, MCE, China), and rabbit anti-beta actin (1:2000, Proteintech, China).

Techniques: Infection, Expressing, Cell Culture, Colorimetric Assay, Quantitative RT-PCR, Control, 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 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: Cellular and Molecular Life Sciences: CMLS

Article Title: Kinesin KIF15 regulates tubulin acetylation and spindle assembly checkpoint in mouse oocyte meiosis

doi: 10.1007/s00018-022-04447-3

Figure Lengend Snippet: KIF15 regulates tubulin acetylation in mouse oocytes. A Western blot analysis of Ac-tubulin expression in KIF15-treated and control oocytes at the MI stage, **, significant difference (P < 0.01). B Ac-tubulin fluorescence staining in control and treated MI stage oocytes. Scale bars, 20 μm. Ac-tubulin is shown in green. C The relative fluorescence intensity of Ac-tubulin in KIF15 treatment groups compared with control group, ***, significant difference (P < 0.001); **, significant difference (P < 0.01). D List of acetylation/deacetylation-related proteins which were correlated with KIF15 by mass spectrometry analysis. E Diagram of STRING analysis showed that Nat10, Sirt2 and Hdac6 interacted with the molecules listed from mass spectrometry analysis. F Co-IP results showed that KIF15 was correlated with the acetylase NAT10 and the deacetylases HDAC6/SIRT2. G Quantitative analysis of the relative intensity of NAT10, SIRT2 and HDAC6 in oocytes by western blot. The results indicated that SIRT2/HDAC6 expression decreased and NAT10 expression increased in KIF15-depleted oocytes. *, significant (P < 0.05)

Article Snippet: The rabbit polyclonal anti-SIRT2 antibody (19655-1-AP, for WB 1:500), anti-NAT10 antibody (13365-a-AP, for WB 1:1,000), anti-HDAC6 antibody (12834-1-AP, for WB 1:1,000), and rabbit monoclonal anti-Bub3 antibody (ET7108-82, for IF 1:100) were purchased from Proteintech (Rocky Hill, NJ, USA).

Techniques: Western Blot, Expressing, Control, Fluorescence, Staining, Mass Spectrometry, Co-Immunoprecipitation Assay