goat nonimmune igg control antibody  (Vector Laboratories)

Journal: The Journal of Investigative Dermatology

Article Title: p63 Transcription Factor Regulates Nuclear Shape and Expression of Nuclear Envelope-Associated Genes in Epidermal Keratinocytes

doi: 10.1016/j.jid.2017.05.013

Figure Lengend Snippet: p63 is enriched at the promoter regions of the Syne3 , Sun1 , and Plec genes. Chromatin isolated from primary mouse keratinocytes was processed for ChIP assay with an antibody against p63 protein or purified mouse IgG. ( a ) Regions within the promoter of Syne3, Sun1 , and Plec analyzed by ChIP-qPCR. Matching p63 core binding site consensus sequences are in red. ( b ) Enrichment of p63 at the Syne3 , Sun1 , and Plec promoter regions. The input levels of unprecipitated chromatin DNA were used as loading controls. Cldn1 and an intergenic region on chr. 8 were used as positive and negative controls, respectively. Error bars represent SD, and four independent experiments were run in triplicates; P < 0.05. ChIP-qPCR, chromatin immunoprecipitation-quantitative PCR; SD, standard deviation.

Article Snippet: Briefly, cross-linked DNA after sonication was precipitated with 5 μg of anti-p63 antibody or nonimmune goat IgG (Vector Laboratories, Burlingame, CA) overnight at 4 °C.

Techniques: Isolation, Purification, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: The Journal of clinical endocrinology and metabolism

Article Title: MUC1 expression is elevated in severe preeclamptic placentas and suppresses trophoblast cell invasion via β1-integrin signaling.

doi: 10.1210/jc.2011-1368

Figure Lengend Snippet: FIG. 6. MUC1 expression inhibits EVT invasion via 1-integrin. A, MUC1 overexpression suppressed cell-ECM adhesion of EVT cells. Mock and MUC1 stable transfectants were seeded into 96-well plates coated with BSA, fibronectin (Fn), collagen IV (Col IV), or laminin (Lam) and incubated at 37 C for 30 min. Adherent cells were counted under a microscope. The number of cells adhered to BSA was subtracted. Representative results obtained from three different fields are shown. Three independent experiments were performed. Error bars represent SD of the mean. *, P 0.05; **, P 0.01. B, Suppression of EVT invasion by MUC1 and functional blocking antibody of 1-integrin. MUC1 stable transfectants showed significant decrease in invasive ability compared with mock transfectants by matrigel invasion assays. The invasion of mock, but not MUC1, transfectants was inhibited by 1-integrin functional blocking antibody (10 g/ml) compared with control IgG. Representative results are shown. Three independent experiments were performed. Error bars represent SD of the mean. **, P 0.01. C, MUC1 overexpression suppressed 1-integrin activity. In the upper panel, active 1-integrins in mock (Ca and Cc) and MUC1 (Cb and Cd) stable transfectants were immunostained by anti-active 1-integrin antibody, followed by FITC-conjugated antimouse antibody. Cell nuclei were stained by DAPI. Images Cc and Cd are amplified pictures from rectangles in Ca and Cb, respectively. Scale bars, 20 m. In the lower panel, flow cytometry with anti-CD29 (TDM29) antibody showed no significant change in the expression of 1-integrins on the cell surface. Negative controls () are mock transfectants stained with secondary antibody alone. D, MUC1 overexpression inhibited phosphorylation of FAK. Mock and MUC1 stable transfectants were preincubated with 1-integrin-blocking antibody or control IgG for 30 min and then seeded into culture plates. Four hours after seeding, cells were collected for immunoblotting with anti-FAK-pY397, anti-total FAK, and ACTB. The ratio of p-FAK and total FAK was quantified (n 3) and shown in the lower panel. **, P 0.01.

Article Snippet: To determine the contribution of 1-integrin to migration and invasion, cells were preincubated with 10 g/ml 1-integrin function-blocking antibody (clone P4C10; Millipore Corporation, Billerica, MA) or 10 g/ml control mouse nonimmune IgG antibody (Southern Biotechnology Associates) for 30 min at 37 C. Immunofluorescence microscopy Cells grown on glass coverslips were fixed for 30 min in 4% paraformaldehyde and preincubated for 15 min in blocking buffer (PBS containing 0.45 mol/liter NaCl, 1% BSA) containing 0.3% (vol/vol) Triton X-100.

Techniques: Expressing, Over Expression, Incubation, Microscopy, Functional Assay, Blocking Assay, Control, Activity Assay, Staining, Amplification, Flow Cytometry, Phospho-proteomics, Western Blot