Journal: Acta biochimica et biophysica Sinica

Article Title: PGAM5 deacetylation mediated by SIRT2 facilitates lipid metabolism and liver cancer proliferation.

doi: 10.3724/abbs.2023155

Figure Lengend Snippet: Figure 2. SIRT2 mediates PGAM5-K191 deacetylation (A) Flag-tagged PGAM5 was stably expressed in PLC cells treated with TSA and/or NAM, followed by immunoprecipitation using an anti-Flag antibody and western blot analysis of the lysine acetylation (K-Ac) of PGAM5. (B) Localization of sirtuin family deacetylases in cells. (C) Immunoprecipitation was performed using anti-Flag antibody or IgG in PLC cells. Flag-PGAM5 and HA- SIRT2 expression were determined by western blot analysis. (D) PLC cells stably expressing Flag-PGAM5 were further transfected with vectors expressing EV or HA-SIRT2. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. The acetylation of Flag-PGAM5 was detected by western blot analysis. (E) PLC cells stably expressing Flag-PGAM5 were further transfected with vectors expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. The acetylation of Flag-PGAM5 was determined by western blot analysis. (F) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with vectors expressing Flag-PGAM5- WT, Flag-PGAM5-K191R and Flag-PGAM5-K191Q. The above cells were subsequently infected with viruses expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. The acetylation of Flag-PGAM5 was detected by western blot analysis. (G) Diagram of the mechanism by which SIRT2 regulates the deacetylation of PGAM5-K191.

Article Snippet: Primary antibodies against the following proteins were used: PGAM5 (Cat# sc-515880; Santa Cruz, Santa Cruz, USA), PGAM5 (Cat# 28445-1-AP; Proteintech, Rosemont, USA), SIRT2 (Cat# 19655-1-AP; Proteintech), SIRT2 (Cat# sc-28298; Santa Cruz), ME1 (Cat# 16619-1-AP; Proteintech), acetylated-lysine (Cat# 9441; CST, Beverly, USA), acetylated-lysine (Cat# PTM-105; Jingjie PTM BioLab, Hangzhou, China), phosphorylation-serine (Cat# sc-81514; Santa Cruz), HA-HRP (Cat# 2999; CST), HA (Cat# 51064-2-AP; Proteintech), mouse IgG1 isotype control (Cat# 66360-1-Ig; Proteintech), Flag (Cat# F1804; Sigma-Aldrich), and β-actin (Cat# 66009-1-Ig; Proteintech).

Techniques: Stable Transfection, Immunoprecipitation, Western Blot, Expressing, Transfection, shRNA, Knockdown, Infection

Journal: Acta biochimica et biophysica Sinica

Article Title: PGAM5 deacetylation mediated by SIRT2 facilitates lipid metabolism and liver cancer proliferation.

doi: 10.3724/abbs.2023155

Figure Lengend Snippet: Figure 3. The deacetylation of PGAM5-K191 mediated by SIRT2 facilitates ME1 dephosphorylation and activity (A) Flag-PGAM5 and HA-ME1 were co-overexpressed in PLC cells. Immunoprecipitation was performed using an anti-Flag antibody or IgG, followed by western blot analysis of HA-ME1 and Flag-PGAM5. (B) PLC cells stably expressing Flag-PGAM5 were further transfected with vectors expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. ME1 and Flag-PGAM5 were determined by western blot analysis. (C) PLC cells stably expressing Flag-PGAM5 were further transfected with empty vector (EV) or vectors expressing HA-SIRT2. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. ME1 and Flag-PGAM5 were determined by western blot analysis. (D) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with EV or vectors expressing Flag-wild-type- PGAM5, Flag-PGAM5-K191R and Flag-PGAM5-K191Q. The above cells were subsequently infected with viruses expressing HA-ME1. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. Flag-PGAM5 and HA-ME1 were determined by western blot analysis. (E) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with EV or vectors expressing HA-PGAM5, HA-PGAM5-K191R and HA-PGAM5-K191Q. The above cells were knocked down with ME1 shRNA and were subsequently infected with viruses expressing Flag-ME1-WT or Flag-ME1-S336A. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. Phosphorylation of ME1, Flag-ME1 and HA-PGAM5 was determined by western blot analysis. (F) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with vectors expressing PGAM5-WT, PGAM5-K191R and PGAM5-K191Q. The above cells were knocked down with ME1 shRNA and were subsequently infected with viruses expressing Flag-ME1-WT or Flag-ME1-S336A. Meanwhile, the cells were infected with the viruses expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in the above cells. Phosphorylation of ME1, Flag-ME1, HA-PGAM5 and SIRT2 was detected by western blot analysis. (G) The metabolic enzyme activity of ME1 was detected by a kit assay in the PLC cells used in (F). The enzyme activity data are expressed as the mean±SD (n=3) and compared with the corresponding groups. *P<0.05, ns, no significant difference.

Article Snippet: Primary antibodies against the following proteins were used: PGAM5 (Cat# sc-515880; Santa Cruz, Santa Cruz, USA), PGAM5 (Cat# 28445-1-AP; Proteintech, Rosemont, USA), SIRT2 (Cat# 19655-1-AP; Proteintech), SIRT2 (Cat# sc-28298; Santa Cruz), ME1 (Cat# 16619-1-AP; Proteintech), acetylated-lysine (Cat# 9441; CST, Beverly, USA), acetylated-lysine (Cat# PTM-105; Jingjie PTM BioLab, Hangzhou, China), phosphorylation-serine (Cat# sc-81514; Santa Cruz), HA-HRP (Cat# 2999; CST), HA (Cat# 51064-2-AP; Proteintech), mouse IgG1 isotype control (Cat# 66360-1-Ig; Proteintech), Flag (Cat# F1804; Sigma-Aldrich), and β-actin (Cat# 66009-1-Ig; Proteintech).

Techniques: De-Phosphorylation Assay, Activity Assay, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Transfection, shRNA, Plasmid Preparation, Knockdown, Infection, Phospho-proteomics

Journal: Innate immunity

Article Title: CCR-2 neutralization augments murine fresh BMC activation by Staphylococcus aureus via two distinct mechanisms: at the level of ROS production and cytokine response.

doi: 10.1177/1753425917697806

Figure Lengend Snippet: Figure 3. Alteration in H2O2, O2 – anion and NO release by murine FBMCs infected with S. aureus after CCR-2 blocking. FBMCs, incubated with or without anti-CCR-2 Abs for 30 min and infected with S. aureus for 30, 60 and 90 min. Supernatant and lysate were prepared as mentioned in the ‘Materials and methods’. H2O2 release in (a) supernatant and (b) lysate, O2 – anion release in (c) supernatant and (d) lysate, and NO release in (e) supernatant and (f) lysate are expressed in terms of M/106 cells. Results are shown as mean SD of three independent experiments. *P < 0.05 compared with uninfected control group; #P < 0.05 with respect to S. aureus-infected group; $P < 0.05 with respect to IgG isotype pre-treated FBMCs prior to S. aureus infection.

Article Snippet: Swiss albino mice were pre-treated with either anti-CCR-2 Ab or control IgG isotype (500mg per mouse) in PBS the day before S. aureus infection.73 We did not find any reference in favor of the dose of the antiCCR-2 Ab purchased from Biorbyt (Cat. No. orb 10487), and we used 500 mg per mouse of this Ab as opposed to the in vivo dosing of the control IgG isotype.

Techniques: Infection, Blocking Assay, Incubation, Control

Journal: Innate immunity

Article Title: CCR-2 neutralization augments murine fresh BMC activation by Staphylococcus aureus via two distinct mechanisms: at the level of ROS production and cytokine response.

doi: 10.1177/1753425917697806

Figure Lengend Snippet: Figure 6. Alteration in cytokine (TNF-a, IFN-g, IL-6, IL-1b, CCL-2 and IL-10) production by murine FBMCs infected with S. aureus after CCR-2 blocking. FBMCs pre-incubated with or without anti-CCR-2 Ab/isotype control Ab for 30 min were infected with S. aureus for the time indicated. Levels of (a) TNF-a, (b) IFN-g, (c) IL-6, (d) IL-1b, (e) CCL-2 (f) and IL-10 in the supernatants were determined by ELISA, according to the manufacturer’s recommendations, and are expressed from triplicate experiments. Values are expressed as mean SD of three independent experiments. *P < 0.05 compared with uninfected control group; #P < 0.05 with respect to S. aureus-infected group; $P < 0.05 with respect to IgG isotype-pre-treated fresh BMCs prior to S. aureus infection.

Article Snippet: Swiss albino mice were pre-treated with either anti-CCR-2 Ab or control IgG isotype (500mg per mouse) in PBS the day before S. aureus infection.73 We did not find any reference in favor of the dose of the antiCCR-2 Ab purchased from Biorbyt (Cat. No. orb 10487), and we used 500 mg per mouse of this Ab as opposed to the in vivo dosing of the control IgG isotype.

Techniques: Infection, Blocking Assay, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: Innate immunity

Article Title: CCR-2 neutralization augments murine fresh BMC activation by Staphylococcus aureus via two distinct mechanisms: at the level of ROS production and cytokine response.

doi: 10.1177/1753425917697806

Figure Lengend Snippet: Figure 7. Expression of CCR-2 receptor by murine fresh bone marrow cells (FBMCs) infected with S. aureus in presence or absence of anti CCR-2 antibody/isotype control antibody Whole cell lysate was prepared for analysis of CCR-2 expression by western blot. All the samples were probed with b-tubulin to show equal protein loading. (a) Lane 1 (FBMC) represented CCR-2 expression on fresh bone marrow cells; lane 2 (FBMC-IgG isotype) represented CCR-2 expression on control IgG isotype treated FBMCs; lane 3 (FBMC- Anti CCR-2 antibody) represented CCR-2 expression on anti CCR-2 antibody treated FBMCs; lane 4 (FBMC+SA) represented CCR- 2 expression on S. aureus infected FBMCS; lane 5 (FBMC+IgG isotype+SA) represented CCR-2 expression on control IgG isotype pretreated FBMC after S. aureus infection and lane 6 (FBMC+ Anti CCR-2 antibody +SA) represented CCR-2 expression on anti CCR-2 antibody pretreated FBMC after S. aureus infection for 30 min, 60 and 90 minute respectively. (b) Fold changes of CCR-2 expression were expressed as mean SD of three independent experiments. ‘*’ indicates significant difference (p < 0.05) compared to uninfected control group; ‘#’ indicates significant difference p < 0.05) with respect to S. aureus infected group; ‘$’ indicates significant difference (p < 0.05) with respect to IgG isotype pretreated fresh bone marrow cells prior to S. aureus infection.

Article Snippet: Swiss albino mice were pre-treated with either anti-CCR-2 Ab or control IgG isotype (500mg per mouse) in PBS the day before S. aureus infection.73 We did not find any reference in favor of the dose of the antiCCR-2 Ab purchased from Biorbyt (Cat. No. orb 10487), and we used 500 mg per mouse of this Ab as opposed to the in vivo dosing of the control IgG isotype.

Techniques: Expressing, Infection, Control, Western Blot

Journal: Innate immunity

Article Title: CCR-2 neutralization augments murine fresh BMC activation by Staphylococcus aureus via two distinct mechanisms: at the level of ROS production and cytokine response.

doi: 10.1177/1753425917697806

Figure Lengend Snippet: Figure 11. Transwell migration assay. Monocyte chemotactic protein-1 (CCL-2) induces in vitro migration of FBMCs. CCL-2 chemotaxis of FBMCs was studied with Transwell Permeable Supports with 10 ng/ml CCL-2 protein (experimental group) or without protein (control group) in the bottom chamber. All cells were seeded on the upper chamber. After the assay was performed, the migratory cells on the bottom side of the membrane were stained and counted. Values are expressed as median SD of three independent experiments. *P < 0.05 compared with uninfected control group; #P < 0.05 with respect to S. aureus-infected group. $P < 0.05 with respect to IgG isotype-pre-treated FBMCs prior to S. aureus infection.

Article Snippet: Swiss albino mice were pre-treated with either anti-CCR-2 Ab or control IgG isotype (500mg per mouse) in PBS the day before S. aureus infection.73 We did not find any reference in favor of the dose of the antiCCR-2 Ab purchased from Biorbyt (Cat. No. orb 10487), and we used 500 mg per mouse of this Ab as opposed to the in vivo dosing of the control IgG isotype.

Techniques: Transwell Migration Assay, In Vitro, Migration, Chemotaxis Assay, Control, Membrane, Staining, Infection

Journal: Cancers

Article Title: Landscape of Bone Marrow Metastasis in Human Neuroblastoma Unraveled by Transcriptomics and Deep Multiplex Imaging

doi: 10.3390/cancers13174311

Figure Lengend Snippet: Validated 20-plex antibody panel and MELC imaging sequence. All primary and secondary antibodies, which passed the validation procedure and were included in the final 20-plex MELC panel. Negative control secondary antibodies were implemented, which were applied to the sample prior to indirect staining of the respective primary antibody. n.r., not relevant.

Article Snippet: 7 , CD3 , PE , monoclonal mouse IgG1 , UCHT1 , ImmunoTools , 21620034 , 1:20.

Techniques: Imaging, Sequencing, Biomarker Discovery, Negative Control, Staining, Recombinant