Journal: Journal of Biological Chemistry

Article Title: Enhanced Deacetylation of p53 by the Anti-apoptotic Protein HSCO in Association with Histone Deacetylase 1

doi: 10.1074/jbc.m609751200

Figure Lengend Snippet: FIGURE 6. Interaction of HSCO with HDAC1. A, co-immunoprecipitation (IP) of exogenous proteins. U-2 OS cells were co-transfected with plasmids expressing HSCO-FLAG and EGFP-HDAC1 as indicated. Cell lysates (10% input) and immunoprecipitates prepared by IP with indicated antibodies were analyzed by Western blotting (WB). Arrows indicate mobilities of specific bands. B, co-IP of HDAC1 and mutant HSCO. U-2 OS cells were co-transfected with plasmids expressing mutant (H79N) HSCO tagged with FLAG and EGFP- HDAC1as indicated and analyzed as in A except that biotinylated antibodies were used in WB. C, co-IP in the absence of p53. H1299 cells were co-trans- fectedwithplasmidsexpressingHSCO-FLAGandHA-HDAC1asindicatedand analyzed as in A. D, co-IP of endogenous proteins. A549 cells were treated with actinomycin D for 0 or 8 h. Cell lysates (10% input) and immunoprecipi- tatespreparedwithanti-HDAC1antibodyorcontrolIgGwereanalyzedbyWB using indicated antibodies. E, GST-pulldown assays. GST-HDAC1 fusion pro- tein, GST-RelA, or GST was incubated with recombinant HSCO, and bound proteins and 10% inputs were analyzed by WB using anti-HSCO antibody (upper panel). GST-HSCO or GST were incubated with recombinant HDAC1, and bound proteins and 10% inputs were analyzed by WB using anti-HDAC1 antibody (lower panel). F, GST-pulldown assay using cell lysates. GST-HSCO or GST was incubated with lysates of U2-OS cells transfected with plasmids expressing FLAG-HDAC1. Bound proteins and 10% inputs were analyzed by WB using anti-FLAG antibody. G, binding in the absence of p53. GST-HSCO or GST was incubated with lysates of H1299 cells transfected with plasmids expressing FLAG-HDAC1 and analyzed as in F. H, co-IP in the absence of p53 and Mdm2. DKO-MEFs were co-transfected with plasmids expressing HSCO- FLAG and EGFP-HDAC1 as indicated and analyzed as in A.

Article Snippet: In some experiments, biotinylated anti-GFP antibody (B-2, Santa Cruz Biotechnology) and anti-FLAG antibody (BioM2, Sigma) were used.

Techniques: Immunoprecipitation, Transfection, Expressing, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Incubation, Recombinant, GST Pulldown Assay, Binding Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Tetravalent Bispecific Tandem Antibodies Improve Brain Exposure and Efficacy in an Amyloid Transgenic Mouse Model

doi: 10.1016/j.omtm.2020.08.014

Figure Lengend Snippet: Bispecific IgG Formats Reported to Enhance Brain Exposure of Proteins (A) Tetravalent bispecific format fusing two Aβ ScFvs to the C-terminal heavy chains of a high affinity TfR antibody. (B) Bivalent high or medium affinity TfR antibody fusing two payload proteins to the C-terminal heavy chains. , , , (C) Tetravalent bispecific format fusing two Aβ ScFvs to the C-terminal light chains of a therapeutic antibody. (D) Brain shuttle format fusing one TfR scFab to one of the IgG heavy chain. (E) Divalent bispecific format. (F) Therapeutic antibody with TfR affinity engineered in its Fc domain. (G) Tribody format linking two anti-Aβ ScFvs to an anti-TfR Fab. (H and I) DVDs and TBTIs . Blue, TfR; green, Aβ; pink, BACE1 recognizing paratopes; yellow, payload proteins such as IL-1 receptor antagonist, TNF-alpha decoy receptor protein, GDNF, or lysosomal enzymes such as iduronate sulfatase.

Article Snippet: They were incubated overnight at room temperature with primary antibodies: biotinylated mouse monoclonal anti-Aβ peptide antibody (4G8, human Aβ17-24, 800705, Biolegend, dilution 1/2,000 or 9240-10, Signet, dilution 1/2,000) or biotinylated goat anti-mouse IgG1 antibody (1070-08, Southern Biotech, dilution 1/200).

Techniques:

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Tetravalent Bispecific Tandem Antibodies Improve Brain Exposure and Efficacy in an Amyloid Transgenic Mouse Model

doi: 10.1016/j.omtm.2020.08.014

Figure Lengend Snippet: Brain Uptake and Distribution of AF-488-Labeled Antibodies in Old APPSL Transgenic Mice Representative images of single green fluorescent of AF-488-labeled administered antibodies (two i.v. injections at 20 mg/kg) or multi-fluorescent emissions over coronal brain sections (counterstained or not with DAPI) of APPSL mice 72 h after second i.v. injection. Green fluorescent signals were restricted to vascular structures with control IgG1-AF488 antibody (A) and anti-Aβ 13C3a-AF488 antibody (B) and predominantly detectable within parenchyma with TBTI3a-AF488 (C) and TBTI6a-AF488 (D). TBTI3a-AF488 administered antibody (E–G) accumulates in parenchymal Aβ deposits as evidenced by comparative analysis of Aβ 4G8 immunolabelling (H–J) over the entire section or both thalamic (single asterisk) and cortical (double asterisks) subareas. Multi-fluorescence emissions confirmed the 13C3a-AF488 green signal was mainly concentrated in vasculature structures of circumventricular areas and parenchymal vessels also stained with Angiospark positive fluorescent signal (pink) (K) and absent over or at proximity of parenchymal Congo red-positive deposits (L). With TBTI3a-AF488 (M and N), marked green signals are surrounding thalamic parenchymal Congo red amyloid deposits, indicative of enhanced brain penetration efficacy as compared to 13C3a administered antibody. The high density of nuclei stained in blue with DAPI around parenchymal Congo red deposits is indicative of characteristic inflammatory response associated with the cerebral Aβ peptide deposition process (L and N). Scale bars, 100 μm (A–D, G, and J) or 50 μm (K–N, F, and I).

Article Snippet: They were incubated overnight at room temperature with primary antibodies: biotinylated mouse monoclonal anti-Aβ peptide antibody (4G8, human Aβ17-24, 800705, Biolegend, dilution 1/2,000 or 9240-10, Signet, dilution 1/2,000) or biotinylated goat anti-mouse IgG1 antibody (1070-08, Southern Biotech, dilution 1/200).

Techniques: Labeling, Transgenic Assay, Injection, Control, Fluorescence, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Tetravalent Bispecific Tandem Antibodies Improve Brain Exposure and Efficacy in an Amyloid Transgenic Mouse Model

doi: 10.1016/j.omtm.2020.08.014

Figure Lengend Snippet: Effect of 13C3a and TBTI Antibodies on Brain Aβ Levels in APPSL Mice Quantification of Aβ38, Aβ40, and Aβ42 in cortical (A–C) and hippocampal (D–F) homogenates of APPSL mice after chronic administration of Ctrl IgG1 (70 nmol/kg), anti-Aβ (13C3a, 70 nmol/kg), or bispecific anti-Aβ/anti-TfR antibodies (TBTI3a and TBTI6a, 15 nmol/kg). Data are means ± SEM. % calculated as means vs Ctrl IgG1. Statistics: one-way ANOVA followed by Newman-Keuls post hoc analysis on Log-transformed data. ∗∗p < 0.01, ∗∗∗p < 0.01 versus Ctrl IgG1, ° p < 0.05 versus 13C3a. No other significant differences between 13C3a and TBTI3a or TBTI6a.

Article Snippet: They were incubated overnight at room temperature with primary antibodies: biotinylated mouse monoclonal anti-Aβ peptide antibody (4G8, human Aβ17-24, 800705, Biolegend, dilution 1/2,000 or 9240-10, Signet, dilution 1/2,000) or biotinylated goat anti-mouse IgG1 antibody (1070-08, Southern Biotech, dilution 1/200).

Techniques: Transformation Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Tetravalent Bispecific Tandem Antibodies Improve Brain Exposure and Efficacy in an Amyloid Transgenic Mouse Model

doi: 10.1016/j.omtm.2020.08.014

Figure Lengend Snippet: Aβ Load and IgG1 Immunostaining in the Cortex and Hippocampus of APPSL Mice Treated with 13C3a and TBTI Antibodies Quantitative analysis of Aβ immunostaining (4G8) in the cortex and hippocampus of APPSL mice after chronic administration of anti-Aβ (13C3a, 70 nmol/kg) or bispecific anti-Aβ/anti-TfR antibodies (TBTI3a and TBTI6a, 15 nmol/kg). Total surface and deposit numbers in the cortex (A and B) and hippocampus (C and D) over 8 cortical rostro-caudal anatomical levels. Data are means ± SEM. % calculated as means vs Ctrl IgG1. Statistics: one-way ANOVA followed by Newman-Keuls post hoc analysis on raw data (for cortex, A and B) or on Log-transformed data (for hippocampus, C and D). ∗∗p < 0.01, ∗∗∗p < 0.001 versus Ctrl IgG1. °p < 0.05, °p < 0.001 versus 13C3a. Representative images of cortical and hippocampal subareas of sagittal hemibrain sections immunostained with anti-IgG1 antibody and counterstained with Congo red of APPSL mice administered once a week for 4 months with Ctrl IgG1 (E), 13C3a (G), or bispecific anti-Aβ/anti-TfR (TBTI3a and TBTI6a) antibodies (I and K). Higher magnification views within cortical fields evidenced that remaining parenchymal Congo red-positive deposits could be decorated with IgG1 immunostaining, indicative that 13C3a (H), TBTI3a (J), and TBTI6a (L) reach their target. As expected, no IgG1 labeling was detected over Congo red-positive deposits after chronic treatment with Ctrl IgG1 antibody (F). Scale bar in the cortical insets, 50 μm.

Article Snippet: They were incubated overnight at room temperature with primary antibodies: biotinylated mouse monoclonal anti-Aβ peptide antibody (4G8, human Aβ17-24, 800705, Biolegend, dilution 1/2,000 or 9240-10, Signet, dilution 1/2,000) or biotinylated goat anti-mouse IgG1 antibody (1070-08, Southern Biotech, dilution 1/200).

Techniques: Immunostaining, Transformation Assay, Labeling

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Tetravalent Bispecific Tandem Antibodies Improve Brain Exposure and Efficacy in an Amyloid Transgenic Mouse Model

doi: 10.1016/j.omtm.2020.08.014

Figure Lengend Snippet: Effect of TBTI Antibodies on TfR Levels in Brain Endothelial Cells and Neurons Expression of murine TfR in bEnd.3 cells (A) and in vitro cultured mouse primary cortical neurons (B) following exposure to TBTI2a, TBTI3a, and TBTI6a versus a control IgG1 for 24 h. TfR protein levels from whole cell lysates were normalized to α-tubulin and compared to control IgG1-treated cells, while TfR protein levels from cell surface lysates were normalized to N-cadherin and compared to control IgG1-treated cells. In both whole cell and surface cell lysates, protein levels were assessed by western blot. Bar graphs show means of each group (±SD); p values were obtained by one-way ANOVA with Dunnet’s multiple comparison test versus Ctrl IgG1 groups. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns p > 0.05.

Article Snippet: They were incubated overnight at room temperature with primary antibodies: biotinylated mouse monoclonal anti-Aβ peptide antibody (4G8, human Aβ17-24, 800705, Biolegend, dilution 1/2,000 or 9240-10, Signet, dilution 1/2,000) or biotinylated goat anti-mouse IgG1 antibody (1070-08, Southern Biotech, dilution 1/200).

Techniques: Expressing, In Vitro, Cell Culture, Control, Western Blot, Comparison

Journal: International immunopharmacology

Article Title: NLRP3 Inflammasome and Glia Maturation Factor Coordinately Regulate Neuroinflammation and Neuronal Loss in MPTP Mouse Model of Parkinson’s Disease

doi: 10.1016/j.intimp.2020.106441

Figure Lengend Snippet: Expression of NLRP3 and Caspase-1 in the PD and non-PD brain SN. Free-floating sections were made from human post-mortem mid brain of the SN (40-μm thickness) and stained using avidin-biotin complex solution. The sections were finally developed by using Novared peroxidase substrate (A). The relative expression of NLRP3 (B) and Caspase-1 (C) were significantly increased in SN of PD brain as compared with SN of non-PD brain. Arrows indicate the NLRP3 stained cells and arrowheads indicate the Caspase-1 stained cells. *p < 0.05 compared to non-PD brain and it was considered as statistically significant. The values are expressed as mean ± SEM (n=5–6/grouop). Magnifications 400X; scale bar = 100 μm.

Article Snippet: The sections were then washed with PBS 3 times and developed using Novared peroxidase substrate (Vector laboratories).

Techniques: Expressing, Staining, Avidin-Biotin Assay

Journal: International immunopharmacology

Article Title: NLRP3 Inflammasome and Glia Maturation Factor Coordinately Regulate Neuroinflammation and Neuronal Loss in MPTP Mouse Model of Parkinson’s Disease

doi: 10.1016/j.intimp.2020.106441

Figure Lengend Snippet: The expression of TH and IL-1β in the SN of MPTP treated mice brain. Free-floating sections were made from MPTP treated midbrain SN (30-μm thickness) and stained with TH (green fluorescence) and IL1β (red fluorescence) (A). After MPTP treatment, there was a qualitative increase in the IL-1β (A). To quantitatively measure the TH immunopositive cells, sections were prestained with TH antibody than stained by using avidin-biotin complex solution. The sections were finally developed by using Novared peroxidase substrate (B and C). The bar graph shows decrease in the number of TH positive neurons in the SN of WT as compared to GMF−/− mice (D). TH immunoreactivity in the striatum of WT mice were significantly reduced than in GMF−/− mice treated with MPTP indicates there is profound expression of TH striatal fibers observed in GMF−/− as compared to WT mice treated with MPTP (C). *p<0.05 compared with MPTP treated WT mice and expressed as number of TH positive cells/407mm2; n=6. Magnification: 400X and 200X. Scale bar = 100 μm.

Article Snippet: The sections were then washed with PBS 3 times and developed using Novared peroxidase substrate (Vector laboratories).

Techniques: Expressing, Staining, Fluorescence, Avidin-Biotin Assay