Journal: Oncogene

Article Title: Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin

doi: 10.1038/onc.2009.206

Figure Lengend Snippet: β4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. A, β4 and β1 integrin expression were determined by Western blot analysis and β4 integrin was also analyzed by Northern blot analysis in 4 osteosarcoma (U2OS, SaOS, G292, and HOS), 2 rhabdomyosarcoma (RD and Rh18), and 2 Ewing’s sarcoma (RDES and TC32) cell lines. B, expression of β4, β1, β3, and α6 integrins was determined by Western blot analysis in the non-metastatic human osteosarcoma cell line HOS and the highly metastatic cell line MNNG-HOS. C, tissue array analysis of β4 integrin expression was performed on human osteosarcoma samples by immunohistochemistry.

Article Snippet: Rabbit polyclonal β4 integrin primary antibody (sc9090, Santa Cruz Biotechnology) at 1:50 dilution was added overnight followed by biotinylated polyclonal goat anti-rabbit immunoglobulin secondary antibody (Dako) at 1:100 dilution for 1 hour.

Techniques: Expressing, Western Blot, Northern Blot, Immunohistochemistry

Journal: Oncogene

Article Title: Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin

doi: 10.1038/onc.2009.206

Figure Lengend Snippet: Knockdown of β4 integrin expression results in a clumpy cell morphology in 3D co-cultures. A, MNNG-HOS (Luc) cells were infected with lentivirus expressing control-shRNA or β4 integrin-shRNA. Expression β4 and α6 integrins was analyzed by Western blot analysis in control-shRNA or β4 integrin-shRNA infected cells. B, the cell surface expression of β4 integrin was evaluated by FACS in control and β4 integrin knockdown cells. Normal IgG was used as a negative control (figure not shown). C, the surface expression and localization of β4 integrin were examined by immunofluorescent staining using same antibody as above in control and knockdown cells. D, MNNG-HOS (Luc) cells infected with lentivirus of control-shRNA (left panel) or β4 integrin-shRNA (right panel) are shown on top as traditional 2D images. Below, are ostoesarcoma cells that were labeled with CellTracker Green CMTPX dye (green). NIH3T3 fibroblasts were labeled separately with CellTracker Orange CMRA dye (red). All cells were then labeled with Hoechst 33342 nuclear dye (blue) and co-cultured in a nanofiber-based matrix labeled with Cy5 dye (white) containing Cultrex basement membrane extract. The lower right panels show merged views.

Article Snippet: Rabbit polyclonal β4 integrin primary antibody (sc9090, Santa Cruz Biotechnology) at 1:50 dilution was added overnight followed by biotinylated polyclonal goat anti-rabbit immunoglobulin secondary antibody (Dako) at 1:100 dilution for 1 hour.

Techniques: Knockdown, Expressing, Infection, Control, shRNA, Western Blot, Negative Control, Staining, Labeling, Cell Culture, Membrane

Journal: Oncogene

Article Title: Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin

doi: 10.1038/onc.2009.206

Figure Lengend Snippet: Control-shRNA and β4 integrin-shRNA infected MNNG-HOS (Luc) cells were injected into the tail vein of RAG knockout mice. These mice then underwent luminescent imaging. Suppression of β4 integrin expression by shRNA led to significant reduction of luminescent intensity in the lungs of mice 50 days after injection of cells, shown graphically (A) with representative lung images at time of sacrifice (B). C, quantitation of luminescence for animals in (A). D, Kaplan-Meier survival curves for MNNG-HOS control-shRNA or β4-shRNA cells.

Article Snippet: Rabbit polyclonal β4 integrin primary antibody (sc9090, Santa Cruz Biotechnology) at 1:50 dilution was added overnight followed by biotinylated polyclonal goat anti-rabbit immunoglobulin secondary antibody (Dako) at 1:100 dilution for 1 hour.

Techniques: Control, shRNA, Infection, Injection, Knock-Out, Imaging, Expressing, Quantitation Assay

Journal: Oncogene

Article Title: Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin

doi: 10.1038/onc.2009.206

Figure Lengend Snippet: Effect of dominant negative β4 integrin expression on primary tumor growth and experimental metastases in vivo . A, disruption of β4 integrin function by transfection of mutant dominant negative β4 integrin. MNNG-HOS cells were transfected with empty vector (EV) and dominant-negative β4 integrin (Y1494F). After batch selection with medium containing G418, cells were lysed and subjected to Western blot analysis of β4 integrin expression (top panel), and immunoprecipititated with β4 integrin antibody overnight followed by Western blot for phospho-tyrosine and β4 integrin (lower panel). B, the cell surface expression of mutant β4 integrin was measured by FACS in mutant β4 integrin (Y1494F) transfected cells. C, empty vector (EV)- and dominant negative β4 integrin (β4-M)-transfected MNNG-HOS (luc) cells were injected into the gastrocnemius muscle of SCID/BEIGE mice and assayed for growth of primary tumors by luminescent imaging (left panel) and quantitation of luminescence signal (right panel). D, empty vector (EV)- and dominant negative β4 integrin (β4M)-transfected MNNG-HOS (luc) cells were injected into the tail vein of RAG knockout mice. Luminescence was quantified weekly with mean intensity for each group plotted over time. E, Kaplan-Meier survival curves showing percent survival for each cell line over time.

Article Snippet: Rabbit polyclonal β4 integrin primary antibody (sc9090, Santa Cruz Biotechnology) at 1:50 dilution was added overnight followed by biotinylated polyclonal goat anti-rabbit immunoglobulin secondary antibody (Dako) at 1:100 dilution for 1 hour.

Techniques: Dominant Negative Mutation, Expressing, In Vivo, Disruption, Transfection, Mutagenesis, Plasmid Preparation, Selection, Western Blot, Injection, Imaging, Quantitation Assay, Knock-Out

Journal: Oncogene

Article Title: Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin

doi: 10.1038/onc.2009.206

Figure Lengend Snippet: β4 integrin interacts with ezrin. A and B, cell lysates were immunoprecipitated (IP) with an anti-ezrin antibody overnight followed by Western blot analysis for β4 and ezrin using the indicated antibodies (WB). C, purified recombinant proteins for BSA, ezrin-N-terminal region (residues 1–297)(Ez-NT), GST, and GST-ezrin-C-terminal region (residues 280–585)(GST-Ez-CT) were immobilized onto glutathione-sepharose beads and incubated overnight with lysates (input) from SaOS cells or in vitro synthesized β4 integrin. The bound fractions were washed and subjected to SDS-PAGE, followed by immunoblotting with anti-β4 integrin antibody.

Article Snippet: Rabbit polyclonal β4 integrin primary antibody (sc9090, Santa Cruz Biotechnology) at 1:50 dilution was added overnight followed by biotinylated polyclonal goat anti-rabbit immunoglobulin secondary antibody (Dako) at 1:100 dilution for 1 hour.

Techniques: Immunoprecipitation, Western Blot, Purification, Recombinant, Incubation, In Vitro, Synthesized, SDS Page

Journal: Oncogene

Article Title: Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin

doi: 10.1038/onc.2009.206

Figure Lengend Snippet: Both ezrin expression and function are required for maintenance of β4 integrin protein expression. A, The expression of ezrin, β1, β3, and β4 integrin, were analyzed by Western blot in K12, K7M2, and K7M2-ezrin antisense cell lines. B, K7M2 and HOS transfected with control (C) or ezrin (Ez) or β4 siRNA. After 3 days, cells were lysed and subjected to Western blot analysis for ezrin and β4 integrin expression. C, β4 integrin expression was analyzed by Western blot in empty vector-GFP and mutant ezrin (T567A) line clones derived from the parental K7M2 cell line. D, K7M2-ezrin antisense cell line clones 13, 1.46 and 1.52 were treated with the proteosome inhibitor, MG132 (50 mM), for 6 h and then analyzed by Western blot for ezrin and β4 integrin expression. E, quantitative RT-PCR analysis for steady-state levels of β4 integrin mRNA in parental K7M2 cells relative to the K7M2-ezrin antisense 1.46 cell line. Samples were normalized to GAPDH mRNA. Mean normalized fold expression is shown from duplicate, independently isolated RNA samples for each cell line.

Article Snippet: Rabbit polyclonal β4 integrin primary antibody (sc9090, Santa Cruz Biotechnology) at 1:50 dilution was added overnight followed by biotinylated polyclonal goat anti-rabbit immunoglobulin secondary antibody (Dako) at 1:100 dilution for 1 hour.

Techniques: Expressing, Western Blot, Transfection, Control, Plasmid Preparation, Mutagenesis, Clone Assay, Derivative Assay, Quantitative RT-PCR, Isolation

Journal: Frontiers in Neuroscience

Article Title: Top-Down Proteomics of Human Saliva Highlights Anti-inflammatory, Antioxidant, and Antimicrobial Defense Responses in Alzheimer Disease

doi: 10.3389/fnins.2021.668852

Figure Lengend Snippet: XIC peak areas values (mean ± SD) normalized on total protein concentration and frequencies of salivary proteins/peptides resulted to levels significantly different between the AD group and HC group.

Article Snippet: All the chemicals and reagents for immunodetection were purchased from Bio-Rad (Hercules, California, United States); the primary mouse monoclonal antibodies (Abs) for α-defensins, cystatin A, cystatin B, S100A8, S100A9, cystatin SN, and thymosin β4 (Tβ4) were purchased from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Protein Concentration

Journal: Frontiers in Neuroscience

Article Title: Top-Down Proteomics of Human Saliva Highlights Anti-inflammatory, Antioxidant, and Antimicrobial Defense Responses in Alzheimer Disease

doi: 10.3389/fnins.2021.668852

Figure Lengend Snippet: Dot-blotting immunodetection of Tβ4 in salivary pools of AD and HC samples (A) and distribution plot of the XIC peak area values of Tβ4 measured by HPLC-ESI-MS (B) in each salivary sample from the AD and HC groups ( ∗ , p < 0.05; ∗∗ , p < 0.01).

Article Snippet: All the chemicals and reagents for immunodetection were purchased from Bio-Rad (Hercules, California, United States); the primary mouse monoclonal antibodies (Abs) for α-defensins, cystatin A, cystatin B, S100A8, S100A9, cystatin SN, and thymosin β4 (Tβ4) were purchased from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Immunodetection