notch 1 mouse rat r d systems af1057 goat polyclonal ab 2153372  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: Mir-34a-5p Mediates Cross-Talk between M2 Muscarinic Receptors and Notch-1/EGFR Pathways in U87MG Glioblastoma Cells: Implication in Cell Proliferation

doi: 10.3390/ijms19061631

Figure Lengend Snippet: Notch-1 Expression in GBM cell lines. Real time RT-PCR and Western blot analysis ( A and B , respectively) for Notch-1 in U87MG and in U251MG cells ( C and D , respectively) cultured in the absence or presence of 100 μM APE for 24 and 48 h. Representative blots are shown from three independent experiments. GAPDH was used as the internal reference protein (* p < 0.05, ** p < 0.01).

Article Snippet: The primary antibodies used were: (1) goat anti-Notch-1 (1:500) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), (2) rabbit anti-Notch-2 (1:200) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), and (3)mouse anti-EGFR (1:500) (Merck Millipore, Vimodrone, Italy).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Mir-34a-5p Mediates Cross-Talk between M2 Muscarinic Receptors and Notch-1/EGFR Pathways in U87MG Glioblastoma Cells: Implication in Cell Proliferation

doi: 10.3390/ijms19061631

Figure Lengend Snippet: Expression of Notch-1 and miR-34a-5p in GBM cell lines and human brain. Real time RT-PCR analysis of miR-34a-5p ( A ) and Notch-1 ( B ) relative expression in U87MG or U251MG cell lines (black bars) compared to human normal brain (white bar). snRNA U6 and 18S were respectively used as the internal standard (** p < 0.01; *** p < 0.001; t -test).

Article Snippet: The primary antibodies used were: (1) goat anti-Notch-1 (1:500) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), (2) rabbit anti-Notch-2 (1:200) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), and (3)mouse anti-EGFR (1:500) (Merck Millipore, Vimodrone, Italy).

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Mir-34a-5p Mediates Cross-Talk between M2 Muscarinic Receptors and Notch-1/EGFR Pathways in U87MG Glioblastoma Cells: Implication in Cell Proliferation

doi: 10.3390/ijms19061631

Figure Lengend Snippet: Analysis of Notch-1/miR-34-5p interaction. ( A ) Analysis of miR-34a-5p expression in U87MG cells, treated with 100 μM APE, by Northern blot (left) and real time RT-PCR (right) (*** p < 0.001 t -test); ( B ) Upper scheme: representation of Luc/Notch reporter construct. MiR is indicated as a thin line, miRNA response element as a thick line. Lower scheme: representation of 3′UTR region (with related sequences) binding mir-34a-5p. Right panel: luciferase activity (Renilla/Firefly ratio) of Notch-1 3′UTR reporter gene in HEK293 cells transfected for 48 h with the mir-34a-5p expressing vector or with empty vector used as control (Ctrl). Data are presented as mean ± SD from at least three different experiments. (*** p < 0.001 t -test); ( C ) mir-34a-5p over-expression after transfection in U87 cells (** p < 0.01 t -test); ( D ) Western blot analysis for Notch-1 levels in U87MG cells transfected with mir-34a-5p.

Article Snippet: The primary antibodies used were: (1) goat anti-Notch-1 (1:500) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), (2) rabbit anti-Notch-2 (1:200) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), and (3)mouse anti-EGFR (1:500) (Merck Millipore, Vimodrone, Italy).

Techniques: Expressing, Northern Blot, Quantitative RT-PCR, Construct, Binding Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Mir-34a-5p Mediates Cross-Talk between M2 Muscarinic Receptors and Notch-1/EGFR Pathways in U87MG Glioblastoma Cells: Implication in Cell Proliferation

doi: 10.3390/ijms19061631

Figure Lengend Snippet: Schematic representation of the Notch-1/EGFR axis in U87 cells. ( A ) In U87 cells, the downregulated expression of mir-34a-5p caused the up-regulation of Notch-1 expression with consequent increased expression of EGFR. The up-regulation of these two receptors causes increased cell proliferation. ( B ) M2 muscarinic receptors up-regulate the expression of mir-34a-5p, which prevents the increased levels of Notch-1. The down-regulation of Notch-1 negatively affects cell proliferation by the down-regulated expression of EGFR.

Article Snippet: The primary antibodies used were: (1) goat anti-Notch-1 (1:500) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), (2) rabbit anti-Notch-2 (1:200) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), and (3)mouse anti-EGFR (1:500) (Merck Millipore, Vimodrone, Italy).

Techniques: Expressing