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: Cancer Research

Article Title: Hypoxia-Regulated Delta-like 1 Homologue Enhances Cancer Cell Stemness and Tumorigenicity

doi: 10.1158/0008-5472.can-09-1605

Figure Lengend Snippet: Figure 2. DLK1 maintains an undifferentiated NB phenotype. A, BE(2)C cells were differentiated with 1 μmol/L RA or 10 μmol/L BrdUrd for 5 d. DLK1, Sox2, c-kit, and CD-133 were detected by Western blot. B, BE(2)C cells were infected with lentivirus expressing shDLK-2H, shDLK-4H, or empty lentiviral vector (ctrl). Left: Infected cells were stained using antibodies against DLK1 (red) or neuron-specific β-tubulin III (green) with Hoechst 33342 as nuclear stain (blue). Magnification, ×200. Right, expression of DLK1 was analyzed by Western blot, and cells with β-tubulin III–positive neurites were enumerated from five to six random fields (columns, mean; bars, SEM; *, P < 0.0001 versus control). C, left, fluorescence-activated cell sorting (FACS)–selected BE(2)C cells expressing the full-length DLK1 (DLK-FL) or empty retroviral vector (ctrl) were cultured for 3 d with or without 1 nmol/L RA and then stained as in B. Magnification, ×200. Right, neurite-positive cells were counted from five to six random fields (columns, mean; bars, SEM; *, P = 0.0001 versus RA-treated control). D, Western blot for CD133 and Notch1 in ER cells expressing the full-length DLK1 with the empty retroviral vector as control (ctrl).

Article Snippet: Western blot was done as described previously (11) with the following antibodies: polyclonal rabbit anti-DLK1 (1:3,000; Chemicon International); anti-Sox2 (1:1,000; Chemicon International); c-kit (1:500; Zymed Laboratories); CD-133 (1:100; Abgent,); Notch1 (1:2,000), HIF-1α (1:2,000), and HIF-2α (1:1,000; Novus Biologicals); proliferating cell nuclear antigen (PCNA; 1:500; Santa Cruz Biotechnology); and phospho–extracellular signal-regulated kinase (ERK) and total ERK (1:1,000; Cell Signaling), or β-actin (1:20,000; Sigma-Aldrich).

Techniques: Western Blot, Infection, Expressing, Plasmid Preparation, Staining, Control, Fluorescence, FACS, Retroviral, Cell Culture

Journal: Open Medicine

Article Title: GPC2 deficiency inhibits cell growth and metastasis in colon adenocarcinoma

doi: 10.1515/med-2022-0421

Figure Lengend Snippet: Prediction of pathways involved in GPC2: (a and b) the genes co-expressed with GPC2 in COAD tissues were enriched in (a) notch signaling and (b) hedgehog signaling and (c) After transfection of shNC, shGPC2 into HCT-8 and SW620 cells, the protein levels of PTCH1, PTCH2, GLI1, NOTCH1, HES1, and DLL4 were detected by Western blotting ( n = 3). KEGG: Kyoto Encyclopedia of Genes and Genomes. ** P < 0.01, *** P < 0.001.

Article Snippet: After separated by using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel, the proteins were transferred onto polyvinylidenefluoride membranes, which were subsequently incubated with 5% skimmed milk at 25°C for 2 h. The membrane was incubated with GPC2 antibody (Biorbyt, catalog number: orb157203), PTCH1 antibody (Biorbyt, catalog number: orb538088), PTCH2 antibody (Biorbyt, catalog number: orb416229), GLI1 antibody (Proteintech, catalog number: 66905-1-Ig), NOTCH1 antibody (Biorbyt, catalog number: orb256723), HES1 antibody (Biorbyt, catalog number: orb36445), DLL4 antibody (Biorbyt, catalog number: orb97478), or glycerine aldehyde phosphate dehydrogenase antibody (Proteintech, catalog number: 10494-1-AP) at 4°C overnight, followed by horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG(H + L) (Proteintech, Catalog number: SA00001-2) or HRP-conjugated goat anti-mouse IgG(H + L) (Proteintech, Catalog number: SA00001-1) at 25°C for 2 h. Finally, Super Sensitive ECL Solution (Beijing Bai’aolaibo Technology Co., Ltd, Catalog number: MT0076) was used to display the results.

Techniques: Transfection, Western Blot