Journal: Scientific Reports

Article Title: Transgenic Eimeria tenella as a vaccine vehicle: expressing TgSAG1 elicits protective immunity against Toxoplasma gondii infections in chickens and mice

doi: 10.1038/srep29379

Figure Lengend Snippet: The levels of IgG 1 and IgG 2a antibodies to TgSAG1 2 weeks after the boost immunization were analysed via ELISA with recombinant TgSAG1 (n = 10).

Article Snippet: The ELISA protocols were conducted as well as in the above description, except the secondary antibodies used in these assays were an HRP-conjugated goat anti-mouse IgG (Proteintech Group Inc., Chicago) and an HRP-conjugated goat anti-mouse IgG 1 or IgG 2a (Proteintech Group Inc., Chicago) for the isotype analyses.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant

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: bioRxiv

Article Title: Hypoimmunogenic human motor neurons induced from iPSCs in vivo substantially ameliorate ALS disease in large animal models

doi: 10.1101/2025.09.03.673895

Figure Lengend Snippet: (A) Schematic illustration of multiplex genetic-editing strategies for engineered HIP-NILB-iPSCs. (B) Indel analysis by Sanger sequencing for B2M , CIITA , IL6ST and TNFRSF1A in HIP-NILB-iPSCs. (C) Real-time PCR analysis of CD47 , CD24 , IgG1-Fc , CTLA4 and PD-L1 in HIP-NILB-iPSCs. (D) Western blot analysis and relative density quantification of B2M, CIITA, IL6ST, TNFR1, CTLA4-Ig, PD-L1, CD24 and CD47 in HIP-NILB-iPSCs of three independent samples. CIITA was detected in NILB-iPSCs and HIP-NILB-iPSCs upon IFN-γ and TNF-α treatment for 48 h. (E) Karyotyping of HIP-NILB-iPSCs. (F) Scatter plot comparison of transcriptomes for HIP-NILB-iPSCs and NILB-iPSCs. The raw counts for each gene were transformed to log10 value, and genes with more than 1 count in each sample were shown. The pluripotent-related genes were labeled in the diagram. Dashed lines depict the 10-fold changes. The R 2 was determined by Pearson’s correlation. The NILB-iPSCs were used as a control. (G) Heatmap showing expression level of stem cells-related and neuron development-related genes together with the enrichment analysis in WT iPSCs and HIP-NILB-iPSCs upon Dox induced for 0.5-, 4- and 7-days. Data are mean ± SEM; p values were determined using a two-tailed, unpaired Student’s t-test ; * p < 0.05; ** p < 0.01.

Article Snippet: The antibodies used were listed as follows: PE anti-human HLA-A,B,C (BioLegend, 311405, 1:20), PE anti-human HLA-DR,DP,DQ (BioLegend, 361716, 1:20), PE Mouse IgG2a, κ Isotype Ctrl (FC) antibody (BioLegend, 400213, 1:20), PE anti-human CD3 (BD Biosciences, 347347,1:50), FITC anti-human CD107a (BD Biosciences,555800,1:100), FITC Mouse IgG1, κ Isotype Control RUO (BD Biosciences, 555748,1:100), APC anti-human CD56 (BioLegend, 362504, 1:20), PE-Cy5 human CD56 (BD Biosciences, 555517, 1:10), APC Mouse IgG1, κ Isotype Ctrl (FC) antibody (BioLegend, 400122, 1:20), PE anti-mouse CD68 (BioLegend, 137014, 1:100), PE Rat IgG2a, κ Isotype Ctrl (BioLegend, 400508, 1:100), CoraLite® Plus 647-conjugated IBA1 monoclonal antibody (Proteintech, CL647-66827,1:200), CoraLite® Plus 647 Mouse IgG2a Isotype Control (Proteintech, CL647-65244,1:200).

Techniques: Multiplex Assay, Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Comparison, Transformation Assay, Labeling, Control, Expressing, Two Tailed Test