primary antibody against e2f3 sc-878  (Santa Cruz Biotechnology)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Shrimp miR-34 from Shrimp Stress Response to Virus Infection Suppresses Tumorigenesis of Breast Cancer

doi: 10.1016/j.omtn.2017.10.016

Figure Lengend Snippet: The Mechanism of Shrimp miR-34 in Inhibiting Breast Cancer Progression (A) The prediction of genes targeted by shrimp miR-34. The seed sequence of miR-34 is underlined. (B) The direct interaction between shrimp miR-34 and its target genes (i.e., CCND1 , CDK6 , CCNE2 , E2F3 , MET , or FOSL1 ) in breast cancer cells (MDA-MB-231). Luciferase activity was normalized to the ratio of firefly and Renilla luciferase activities. (C) The effects of shrimp miR-34 expression on the expression of miR-34 target genes in breast cancer cells (MDA-MB-231 and MDA-MB-435). (D) The influence of shrimp miR-34 expression on the expression of miR-34 target gene-encoding proteins in breast cancer cells. Protein levels were detected using western blot analysis. As a control, miR-34-scrambled was included in the transfection. Tubulin was used as a loading control. (E) The loading of shrimp miR-34 onto the human Ago2 complex. Shrimp miR-34 was incubated with the human Ago2 complex, followed by EMSA. Shrimp miR-34 was visualized by ethidium bromide staining (top). Human Ago2 protein was detected with western blot analysis (bottom). The wedge indicates the concentration gradient of human Ago2 complex. (F) A model for the role of miR-34-mediated cancer cell growth and metastasis. In all panels, statistically significant differences between treatments are represented with asterisks (error bar, SD; *p < 0.05 and **p < 0.01).

Article Snippet: The paraffin sections were then incubated with primary antibodies against human CCND1, CDK6, CCNE2, E2F3, or MET (Proteintech Group, USA) at 4°C overnight, followed by incubation with HRP-conjugated goat anti-Rabbit IgG (Sigma-Aldrich, USA) for 2 hr at room temperature.

Techniques: Sequencing, Luciferase, Activity Assay, Expressing, Western Blot, Control, Transfection, Incubation, Staining, Concentration Assay

Journal: Molecular Medicine Reports

Article Title: miR-34a suppresses proliferation and induces apoptosis of human lens epithelial cells by targeting E2F3

doi: 10.3892/mmr.2016.5901

Figure Lengend Snippet: E2F3 was a direct target of miR-34a. (A and B) Schematic representation of WT and mut putative miR-34a-binding sites in the 3′-UTR of E2F3. (C) The 293T cells were transfected with miR-34a mimic and psiCHECK2 containing either the WT or mut putative binding sites for miR-34a, and 48 h later the luciferase assay was performed. The data indicated that miR-34a mimic reduced the Renilla activity in the reporter construct containing the E2F3 site (1.87±0.13), whereas no effect was observed with a construct containing a mut E2F3 seed site (3.46±0.09) and mimic control (3.27±0.04). Data are presented as the mean ± standard deviation; *P<0.05. miR, microRNA; WT, wild-type; mut, mutant; UTR, untranslated region; NS, non-significant.

Article Snippet: Subsequent to blocking in Tris-buffered saline with Tween-20 (TBST) containing 25 mmol/l Tris-HCl, pH 7.5, 137 mmol/l NaCl, 2.7 mmol/l KCl and 0.05% Tween-20 with 5% nonfat milk for 1 h at 37°C, the membranes were incubated with the primary antibody against E2F3 (sc-878; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or GAPDH (ab9485; 1:2,500; Abcam, Cambridge, MA, USA) in TBST with 5% nonfat milk at 4°C overnight.

Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Construct, Control, Standard Deviation, Mutagenesis

Journal: Molecular Medicine Reports

Article Title: miR-34a suppresses proliferation and induces apoptosis of human lens epithelial cells by targeting E2F3

doi: 10.3892/mmr.2016.5901

Figure Lengend Snippet: miR-34a and siE2F3 reduce E2F3 expression. SRA01/04 cells transfected with miR-34a mimic or siE2F3 exhibited reduced E2F3 expression compared with the mock and mimic control. Data are presented as the mean ± standard deviation; *P<0.05. miR, microRNA; si, small interfering; NS, non-significant.

Article Snippet: Subsequent to blocking in Tris-buffered saline with Tween-20 (TBST) containing 25 mmol/l Tris-HCl, pH 7.5, 137 mmol/l NaCl, 2.7 mmol/l KCl and 0.05% Tween-20 with 5% nonfat milk for 1 h at 37°C, the membranes were incubated with the primary antibody against E2F3 (sc-878; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or GAPDH (ab9485; 1:2,500; Abcam, Cambridge, MA, USA) in TBST with 5% nonfat milk at 4°C overnight.

Techniques: Expressing, Transfection, Control, Standard Deviation