Journal: Frontiers in Pharmacology

Article Title: Sirt3 Mediates the Inhibitory Effect of Adjudin on Astrocyte Activation and Glial Scar Formation following Ischemic Stroke

doi: 10.3389/fphar.2017.00943

Figure Lengend Snippet: Notch1 signaling pathway was involved in Sirt3-mediated inhibitory effect of adjudin on wound healing in vitro . (A) Western blot showed Notch1 and N1ICD protein levels were decreased by adjudin in WT astrocytes, and no significantly differences were observed in Sirt3 KO astrocytes. (B) Luciferase experiment showed adjudin significantly inhibited the transcriptional activity of N1ICD. (C) To study the role of Notch1 signaling pathway, we transfected N1ICD plasmid into C8-D1A cells. Western blot indicated N1ICD protein level. (D) We performed wound healing experiments performed in C8-D1A cells, and we observed gap area 24 h after scratch. (E) The gap area of C8-D1A at 0 h after scratch. (F) The gap area of C8-D1A at 0 h after scratch. The quantification of gap area in C8-D1A cells showed N1ICD overexpression blunted the inhibitory of adjudin. one-way ANOVA: * P < 0.05, *** P < 0.001, ns: no significance. Bars represent the mean ± SEM. Scale bar, (D) 500 μm.

Article Snippet: The N1ICD plasmid (#41730) and 4xCSL luciferase reporter (#41726) were purchased from Addgene. pRL-TK reference plasmid was provided by Pro.

Techniques: In Vitro, Western Blot, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression

Journal: Frontiers in Pharmacology

Article Title: Sirt3 Mediates the Inhibitory Effect of Adjudin on Astrocyte Activation and Glial Scar Formation following Ischemic Stroke

doi: 10.3389/fphar.2017.00943

Figure Lengend Snippet: Sirt3 mediated the inhibitory effect of adjudin on glial scar formation following ischemic stroke. Mice were treated with either DMSO or adjudin 2 days after tMCAO. (A) The formation of glial scar were inhibited by adjudin in WT mice 35 days after tMCAO, and Sirt3 deficiency blunted the effect of adjudin. (B) The quantification of the maximal thickness of glial scar. (C) The quantification of GFAP protein and mRNA expression. (D) Western blot showed adjudin increased Sirt3 and Foxo3a protein levels and decreased Notch1 and N1ICD protein levels in WT mice 35 days after tMCAO, which were blunted by Sirt3 deficiency. (E) The quantification of protein expression. (F) Rotarod test showed adjudin increased latency time in WT mice, and Sirt3 deficiency blunted the effect. One-way ANOVA: * P < 0.05, ** P < 0.01, ns: no significance ( n = 5 mice per group). Bars represent the mean ± SEM. Scale bar, (A) 100 μm.

Article Snippet: The N1ICD plasmid (#41730) and 4xCSL luciferase reporter (#41726) were purchased from Addgene. pRL-TK reference plasmid was provided by Pro.

Techniques: Expressing, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: USP7 deubiquitinates and stabilizes NOTCH1 in T-cell acute lymphoblastic leukemia

doi: 10.1038/s41392-018-0028-3

Figure Lengend Snippet: USP7 interacts with ICN1. a HEK293T cells were transfected with plasmids encoding FLAG-tagged USP7 and/or Myc-tagged ICN1. Cell extracts were prepared and immunoprecipitated with anti-FLAG or anti-Myc antibodies. The protein interactions were analyzed by western blotting. b Whole-cell lysates from JURKAT and MOLT-4 cells were subjected to immunoprecipitation with a control IgG or an anti-ICN1 antibody. The immunoprecipitates were detected by western blotting. The input represented ~5% of the total protein extract used for immunoprecipitation. c The direct interaction between USP7 and ICN1 was detected using a GST pull-down assay, and the indicated proteins were examined by western blotting. d USP7 was co-localized with NOTCH1. CUTLL1 cells were fixed and immunostained with anti-USP7 (green) and anti-NOTCH1 (red) antibodies. The cell nuclei were counterstained with DAPI (blue). e Mapping of the ICN1-interacting domain in the USP7 protein. Top panel, a schematic representation of various USP7 truncated mutants. Bottom panel, HEK293T cells were co-transfected with constructs encoding FLAG-tagged ICN1 and GFP-tagged USP7 or truncated mutants. FLAG-tagged ICN1 proteins were immunoprecipitated with an anti-FLAG antibody, and the presence of USP7 protein and truncated mutants was examined by western blotting using an anti-GFP antibody

Article Snippet: The following antibodies were used in this study: anti-cleaved NOTCH1 (Val1744) and anti-β-Actin (CST, Danves, MA, USA); anti-USP7 (Bethyl Laboratories, Montgomery, TX, USA); anti-NOTCH1 and anti-GFP (Santa Cruz, Dallas, TX, USA); anti-HA and anti-Myc epitope tag (MBL, Nagoya, Japan); anti-FLAG (M2) (Sigma-Aldrich, Louis, MO, USA); and anti-HRP-conjugated secondary antibody (Millipore, Bedford, MA, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Pull Down Assay, Construct

Journal: Signal Transduction and Targeted Therapy

Article Title: USP7 deubiquitinates and stabilizes NOTCH1 in T-cell acute lymphoblastic leukemia

doi: 10.1038/s41392-018-0028-3

Figure Lengend Snippet: USP7 is overexpressed in T-ALL. a USP7 microarray gene expression data were obtained from the Cancer Cell Line Encyclopedia (CCLE). The data are presented with box plots. The sample number ( n ) are indicated in parentheses. RMA represents Robust Multi-array Average. b Analysis of TCGA leukemia dataset from the Oncomine database to assess the expression of USP7 in normal bone marrow or peripheral blood cells and in T-ALL patient samples. The data are presented with box plots. Fold change, p- value (determined by Student’s t -test), and sample size are shown. c Comparison of USP7 gene expression levels between NOTCH1 WT ( n = 31) and NOTCH1 mutated ( n = 87) T-ALL cases. The read counts mapped to the USP7 transcript were normalized and applied with variance-stabilizing transformation. The p- value was determined using Student’s t -test. Dots represent the value of the USP7 expression level in each of the T-ALL cases. The mean and 25th and 75th percentiles are represented by the midline and line edges in the plots, respectively. d western blotting analysis of the USP7 protein levels in normal PBMCs and T-ALL patient samples (top panel) along with various T-ALL cell lines (bottom panel)

Article Snippet: The following antibodies were used in this study: anti-cleaved NOTCH1 (Val1744) and anti-β-Actin (CST, Danves, MA, USA); anti-USP7 (Bethyl Laboratories, Montgomery, TX, USA); anti-NOTCH1 and anti-GFP (Santa Cruz, Dallas, TX, USA); anti-HA and anti-Myc epitope tag (MBL, Nagoya, Japan); anti-FLAG (M2) (Sigma-Aldrich, Louis, MO, USA); and anti-HRP-conjugated secondary antibody (Millipore, Bedford, MA, USA).

Techniques: Microarray, Expressing, Transformation Assay, Western Blot

Journal: The Journal of cell biology

Article Title: Aberrant phosphorylation inactivates Numb in breast cancer causing expansion of the stem cell pool.

doi: 10.1083/jcb.202112001

Figure Lengend Snippet: Figure 3. Characterization of Numb phosphomutants. (A) Scheme showing the behavior of a SC (A) and daughter progenitor cell (B) in WT vs. Numb-KO (Tosoni et al., 2015). (B) Scheme of the growth of MS from WT and Numb-KO MECs (Tosoni et al., 2015). (C and D) WT and Numb-KO cells, transduced with the indicated constructs (DsRed fusion proteins; EV, empty vector), were assessed for SFE (C, by counting only red cells or MS) and size (D, N = number of epifluorescent MS analyzed). Results are expressed relative to WT cells (see also Table S2). Significance was calculated vs. EV cells. Representative images of the MS are in D, top panel. Bar, 100 µm. (E) WT and Numb-KO MS, transduced with the indicated constructs (Flag-tagged), were analyzed by IB. Arrows, endogenous (black) or overexpressed (red) Numb (also in G). Activated Notch (Act. Notch) was detected with the anti Val1744 Ab (in this and all subsequent figures). Right: Quantitation of three independent experiments. (F) HEK-293 cells, transfected as indicated (all Numb constructs were Flag-tagged and also codify for an sh-RNA sequence against endogenous Numb; EV, empty vector), were IP and IB as shown. (G) HEK-293 cells were stably transduced with Notch- NΔE (Notch-TFX; NT, not transfected) and transfected with the indicated Numb-Flag constructs (as in F). IP and IB were as shown. (H and I) MCF-7 or Cal51 cells were either transduced with Notch-NΔE (Notch-TFX; I) or not (H). Cells were treated with BIS (or mock-treated) and IP and IB as shown. In H, IP-Ctr is anti-Flag; in I, IP-Ctr is goat IgG. Data are reported ± SD (C and E) or ± SE (D). Statistical analysis was with the Student’s t test two-tailed (C and D) or with the one-sample t test (E). Source data are available for this figure: SourceData F3.

Article Snippet: Ab for immunoblot (IB) were directed against Numb (AB21, a mouse monoclonal Ab against amino acids 537–551 of hNumb [Colaluca et al., 2008]), and for the experiment in Fig. S3 B, the anti-Numb C29G11, rabbit monoclonal from Cell Signaling Technologies (Cat. 4140) was used; Vinculin (mouse monoclonal; Sigma-Aldrich, Cat. V9131); GRP94 (9G10, rat monoclonal, Cat. ADI-SPA-851; Enzo Life Sciences); Tubulin (11H10, rabbit monoclonal, Cat. 2125; Cell Signaling Technologies); p53 (1C12, mouse monoclonal, Cat. 2524; Cell Signaling Technologies, Figs. 3 E, 5, A–C, and 9 D; FL393, Santa Cruz Biotechnology, goat polyclonal, Cat. sc-6243-G, Fig. 3 F; goat polyclonal, Bio-techne, Cat. AF1355 Fig. 3 H); PKCζ (C24E6, rabbit monoclonal, Cat. 9368; Cell Signaling Technologies); p-PKCζ (H-2, mouse monoclonal, Cat. sc-271962; Santa Cruz Biotechnology); PAN-PKC (A-9, mouse monoclonal, Cat. sc-17804; Santa Cruz Biotechnology); pPAN PKC (rabbit polyclonal, Cat. 9371; Cell Signaling Technologies); Notch (5B5, rat monoclonal, Cat. 3447; Cell Signaling Technologies); Cleaved Notch1 Val1744 (D3B8, rabbit monoclonal, Cat. 4147; Cell Signaling Technologies); Flag (D6W5B, rabbit monoclonal, Cat. 2368; Cell Signaling Technologies); Par3 (Cat. 07-330; Millipore); Mdm2 (Mouse Monoclonal, Cat. Op46; Calbiochem); anti GAPDH (rabbit monoclonal, Cat. 5174; Cell Signaling Technologies); and pNumb-Ser276 (rabbit monoclonal, Cat. 4140; Cell Signaling Technologies).

Techniques: Transduction, Construct, Plasmid Preparation, Quantitation Assay, Transfection, Sequencing, Stable Transfection, Two Tailed Test

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Fully human monoclonal antibody targeting activated ADAM10 on colorectal cancer cells

doi: 10.1016/j.biopha.2023.114494

Figure Lengend Snippet: 1H5 inhibits Notch cleavage. Sandwich ELISA was used to measure the levels of total (A) and cleaved (B) Notch1 in COLO205 cells upon treatment with 1H5. The data represent mean of triplicate experiments, and the bar plots show the effect of treatment with 1H5 relative to untreated control, mean ± SEM. Comparison of notch levels between treated and untreated groups was performed using independent t test. Total Notch1 levels did not significantly differ between the two groups (A), p = 0.162. On the other hand, the mAb-treated group showed significant decrease of the cleaved Notch1 levels when compared with the untreated control (B), p < 0.001.

Article Snippet: To detect NICD1, we used PathScan ® cleaved Notch1 (Val1744) sandwich ELISA Kit (Cell Signaling Technologies).

Techniques: Sandwich ELISA, Control, Comparison