primary goat antibodies against pdcd4  (Santa Cruz Biotechnology)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: Differential Levels of H19, miR-675, miR-200a, IGF1R, and PDCD4 after MCT Treatment in PAH Rats (A) The H19 level in the MCT plus melatonin group was much higher than in the MCT group, while it was even higher in the control group than in the MCT plus melatonin group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (B) The miR-200a level in the MCT plus melatonin group was much higher than in the control group, while it was even higher in the MCT group than in the MCT plus melatonin group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (C) The miR-675-3p level in the MCT group was much lower than in the MCT plus melatonin group, and both of them were much lower than in the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (D) The IGF1R mRNA level in the MCT group was much higher than in the MCT plus melatonin group, and both of them were much higher than in the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (E) The MCT plus melatonin group displayed a lower level of PDCD4 mRNA than did the control group, while the MCT group exhibited an even lower level of PDCD4 mRNA than did the MCT plus melatonin group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (F) The IGF1R protein level in the MCT group was much higher than in the MCT plus melatonin group, and both of them were much higher than in the control group. The PDCD4 protein level in the MCT group was much lower than in the MCT plus melatonin group, and both of them were much lower than in the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group).

Article Snippet: Subsequently, the sections were blocked with 3% H 2 O 2 to inactivate the activity of endogenous peroxidase, followed by incubation at room temperature for 2 hr with primary goat antibodies against PDCD4 (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and IGF1R (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: Immunohistochemistry for PDCD4 According to the IHC result, the PDCD4 protein level in the MCT group was much lower than in the MCT plus melatonin group, and both of them were much lower than in the control group (scale bars, 10 μm).

Article Snippet: Subsequently, the sections were blocked with 3% H 2 O 2 to inactivate the activity of endogenous peroxidase, followed by incubation at room temperature for 2 hr with primary goat antibodies against PDCD4 (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and IGF1R (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunohistochemistry, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: miR-675-3p and miR-200a Directly Targeted IGF1R and PDCD4, Respectively (A) The sequence comparison between mature miR-675-3p and wild-type as well as mutant IGF1R 3′ UTR. (B) Luciferase activity of wild-type IGF1R 3′ UTR, but not that of mutant IGF1R 3′ UTR, in hPASMCs transfected with miR-675-3p mimic was much lower than scramble control (*p < 0.05 as compared with the control group). (C) Luciferase activity of wild-type IGF1R 3′ UTR, but not that of mutant IGF1R 3′ UTR, in rPASMCs transfected with miR-675 mimic was much lower than scramble control (*p < 0.05 as compared with the control group). (D) The sequence comparison between mature miR-200a and wild-type as well as mutant PDCD4 3′ UTR. (E) Luciferase activity of wild-type PDCD4 3′ UTR, but not that of mutant PDCD4 3′ UTR, in hPASMCs transfected with miR-675 mimic was downregulated compared with scramble control (*p < 0.05 as compared with the control group). (F) Transfecting with miR-200a mimic reduced luciferase activity of wild-type PDCD4 3′ UTR, but not that of mutant PDCD4 3′ UTR, in rPASMCs (*p < 0.05 as compared with the control group).

Article Snippet: Subsequently, the sections were blocked with 3% H 2 O 2 to inactivate the activity of endogenous peroxidase, followed by incubation at room temperature for 2 hr with primary goat antibodies against PDCD4 (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and IGF1R (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Sequencing, Comparison, Mutagenesis, Luciferase, Activity Assay, Transfection, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: Effect of Melatonin on Cell Proliferation and H19, miR-675-3p, miR-200a, IGF1R, and PDCD4 Levels in hPASMCs (A) Melatonin inhibited cell viability of hPASMCs in a dose-dependent manner (*p < 0.05 as compared with the control group). (B) Melatonin inhibited cell viability of hPASMCs in a dose-dependent fashion. (C) Treating with melatonin dose-dependently upregulated H19 expression (*p < 0.05 as compared with the control group). (D) Treating with melatonin dose-dependently enhanced miR-675-3p expression (*p < 0.05 as compared with the control group). (E) miR-200a level was dose-dependently reduced following treatment with melatonin (*p < 0.05 as compared with the control group). (F) IGF1R mRNA level was dose-dependently suppressed after the administration of melatonin (*p < 0.05 as compared with the control group). (G) PDCD4 mRNA level was dose-dependently upregulated subsequent to treatment with melatonin (*p < 0.05 as compared with the control group). (H) IGF1R protein level was dose-dependently suppressed while PDCD4 protein expression was dose-dependently enhanced following treatment with melatonin.

Article Snippet: Subsequently, the sections were blocked with 3% H 2 O 2 to inactivate the activity of endogenous peroxidase, followed by incubation at room temperature for 2 hr with primary goat antibodies against PDCD4 (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and IGF1R (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: Effect of Melatonin on Cell Proliferation and H19, miR-675, miR-200a, IGF1R, and PDCD4 Levels in rPASMCs (A) Melatonin inhibited cell viability of rPASMCs in a dose-dependent manner (*p < 0.05 as compared with the control group). (B) Melatonin inhibited cell viability of rPASMCs in a dose-dependent fashion. (C) Treating with melatonin dose-dependently upregulated H19 expression (*p < 0.05 as compared with the control group). (D) Treating with melatonin dose-dependently enhanced miR-675-3p expression (*p < 0.05 as compared with the control group). (E) miR-200a level was dose-dependently reduced following treatment with melatonin (*p < 0.05 as compared with the control group). (F) IGF1R mRNA level was dose-dependently suppressed after the administration of melatonin (*p < 0.05 as compared with the control group). (G) PDCD4 mRNA level was dose-dependently upregulated subsequent to treatment with melatonin (*p < 0.05 as compared with the control group). (H) IGF1R protein level was dose-dependently suppressed while PDCD4 protein expression was dose-dependently enhanced following treatment with melatonin.

Article Snippet: Subsequently, the sections were blocked with 3% H 2 O 2 to inactivate the activity of endogenous peroxidase, followed by incubation at room temperature for 2 hr with primary goat antibodies against PDCD4 (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and IGF1R (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: Two Signaling Pathways Are Shown Dual signaling pathways (H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4) were involved in the mechanisms underlying the therapeutic effect of melatonin in the treatment of PAH by promoting the apoptosis of PASMCs.

Article Snippet: Subsequently, the sections were blocked with 3% H 2 O 2 to inactivate the activity of endogenous peroxidase, followed by incubation at room temperature for 2 hr with primary goat antibodies against PDCD4 (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and IGF1R (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Protein-Protein interactions

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: Differential Levels of H19, miR-675, miR-200a, IGF1R, and PDCD4 after MCT Treatment in PAH Rats (A) The H19 level in the MCT plus melatonin group was much higher than in the MCT group, while it was even higher in the control group than in the MCT plus melatonin group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (B) The miR-200a level in the MCT plus melatonin group was much higher than in the control group, while it was even higher in the MCT group than in the MCT plus melatonin group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (C) The miR-675-3p level in the MCT group was much lower than in the MCT plus melatonin group, and both of them were much lower than in the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (D) The IGF1R mRNA level in the MCT group was much higher than in the MCT plus melatonin group, and both of them were much higher than in the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (E) The MCT plus melatonin group displayed a lower level of PDCD4 mRNA than did the control group, while the MCT group exhibited an even lower level of PDCD4 mRNA than did the MCT plus melatonin group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (F) The IGF1R protein level in the MCT group was much higher than in the MCT plus melatonin group, and both of them were much higher than in the control group. The PDCD4 protein level in the MCT group was much lower than in the MCT plus melatonin group, and both of them were much lower than in the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group).

Article Snippet: Subsequently, the membrane was incubated at 4°C for 12 hr with anti-β-actin monoclonal antibodies (1:8,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal goat primary antibodies against PDCD4 and IGF1R (1:5,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: Immunohistochemistry for IGF1R According to the IHC result, the IGF1R protein level in the MCT group was much higher than in the MCT plus melatonin group, and both of them were much higher than in the control group (scale bars, 10 μm).

Article Snippet: Subsequently, the membrane was incubated at 4°C for 12 hr with anti-β-actin monoclonal antibodies (1:8,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal goat primary antibodies against PDCD4 and IGF1R (1:5,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunohistochemistry, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: miR-675-3p and miR-200a Directly Targeted IGF1R and PDCD4, Respectively (A) The sequence comparison between mature miR-675-3p and wild-type as well as mutant IGF1R 3′ UTR. (B) Luciferase activity of wild-type IGF1R 3′ UTR, but not that of mutant IGF1R 3′ UTR, in hPASMCs transfected with miR-675-3p mimic was much lower than scramble control (*p < 0.05 as compared with the control group). (C) Luciferase activity of wild-type IGF1R 3′ UTR, but not that of mutant IGF1R 3′ UTR, in rPASMCs transfected with miR-675 mimic was much lower than scramble control (*p < 0.05 as compared with the control group). (D) The sequence comparison between mature miR-200a and wild-type as well as mutant PDCD4 3′ UTR. (E) Luciferase activity of wild-type PDCD4 3′ UTR, but not that of mutant PDCD4 3′ UTR, in hPASMCs transfected with miR-675 mimic was downregulated compared with scramble control (*p < 0.05 as compared with the control group). (F) Transfecting with miR-200a mimic reduced luciferase activity of wild-type PDCD4 3′ UTR, but not that of mutant PDCD4 3′ UTR, in rPASMCs (*p < 0.05 as compared with the control group).

Article Snippet: Subsequently, the membrane was incubated at 4°C for 12 hr with anti-β-actin monoclonal antibodies (1:8,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal goat primary antibodies against PDCD4 and IGF1R (1:5,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Sequencing, Comparison, Mutagenesis, Luciferase, Activity Assay, Transfection, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: Effect of Melatonin on Cell Proliferation and H19, miR-675-3p, miR-200a, IGF1R, and PDCD4 Levels in hPASMCs (A) Melatonin inhibited cell viability of hPASMCs in a dose-dependent manner (*p < 0.05 as compared with the control group). (B) Melatonin inhibited cell viability of hPASMCs in a dose-dependent fashion. (C) Treating with melatonin dose-dependently upregulated H19 expression (*p < 0.05 as compared with the control group). (D) Treating with melatonin dose-dependently enhanced miR-675-3p expression (*p < 0.05 as compared with the control group). (E) miR-200a level was dose-dependently reduced following treatment with melatonin (*p < 0.05 as compared with the control group). (F) IGF1R mRNA level was dose-dependently suppressed after the administration of melatonin (*p < 0.05 as compared with the control group). (G) PDCD4 mRNA level was dose-dependently upregulated subsequent to treatment with melatonin (*p < 0.05 as compared with the control group). (H) IGF1R protein level was dose-dependently suppressed while PDCD4 protein expression was dose-dependently enhanced following treatment with melatonin.

Article Snippet: Subsequently, the membrane was incubated at 4°C for 12 hr with anti-β-actin monoclonal antibodies (1:8,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal goat primary antibodies against PDCD4 and IGF1R (1:5,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: Effect of Melatonin on Cell Proliferation and H19, miR-675, miR-200a, IGF1R, and PDCD4 Levels in rPASMCs (A) Melatonin inhibited cell viability of rPASMCs in a dose-dependent manner (*p < 0.05 as compared with the control group). (B) Melatonin inhibited cell viability of rPASMCs in a dose-dependent fashion. (C) Treating with melatonin dose-dependently upregulated H19 expression (*p < 0.05 as compared with the control group). (D) Treating with melatonin dose-dependently enhanced miR-675-3p expression (*p < 0.05 as compared with the control group). (E) miR-200a level was dose-dependently reduced following treatment with melatonin (*p < 0.05 as compared with the control group). (F) IGF1R mRNA level was dose-dependently suppressed after the administration of melatonin (*p < 0.05 as compared with the control group). (G) PDCD4 mRNA level was dose-dependently upregulated subsequent to treatment with melatonin (*p < 0.05 as compared with the control group). (H) IGF1R protein level was dose-dependently suppressed while PDCD4 protein expression was dose-dependently enhanced following treatment with melatonin.

Article Snippet: Subsequently, the membrane was incubated at 4°C for 12 hr with anti-β-actin monoclonal antibodies (1:8,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal goat primary antibodies against PDCD4 and IGF1R (1:5,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

doi: 10.1016/j.omtn.2018.08.015

Figure Lengend Snippet: Two Signaling Pathways Are Shown Dual signaling pathways (H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4) were involved in the mechanisms underlying the therapeutic effect of melatonin in the treatment of PAH by promoting the apoptosis of PASMCs.

Article Snippet: Subsequently, the membrane was incubated at 4°C for 12 hr with anti-β-actin monoclonal antibodies (1:8,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal goat primary antibodies against PDCD4 and IGF1R (1:5,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Protein-Protein interactions

Journal: Scientific Reports

Article Title: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

doi: 10.1038/srep17528

Figure Lengend Snippet: Hepatocytes were isolated and plated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. ( A ) Real-Time RT-PCR analysis of miR-21 (n = 7). ( B ) Immunoblotting of PDCD4 (top; n = 7) and ratio between Wt and Mut miR-21 luciferase activity (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. Cells were co-transfected with a reporter vector consisting of a luciferase cDNA fused to the 3′ UTR of PDCD4, containing either a Wt or Mut miR-21 binding site. The cytomegalovirus- Renilla luciferase vector was used as an internal standard control. ( C ) Cell viability, measured by the ApoTox-Glo TM Triplex assay (top; n = 5), cell death measured by the LDH assay (middle; n = 7) and caspase-3/7 activity (bottom; n = 5). ( D ) Apoptotic cells were detected by Hoechst staining. Representative images of control and 25, 50, 100 and and 200 μM DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.

Article Snippet: Upon transference onto a nitrocellulose membranes and blocking with 5% milk solution, membranes were probed overnight at 4 °C with primary rabbit polyclonal antibodies against IκB (sc-371), NF-κB p65 (sc-372) and PIDD (sc-67032) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal antibody against active caspase-2 (ab2251; Abcam PLC, Cambridge, United Kingdom), primary goat polyclonal antibody against PDCD4 (sc-27123; Santa Cruz Biotechnology) or primary mouse monoclonal antibodies against β-actin (A5441; Sigma-Aldrich Co.), HDAC1 (05–100; Upstate Biotechonology, Lake Placid, NY, USA), GAPDH (sc-32233; Santa Cruz Biotechnology) and, finally, with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature.

Techniques: Isolation, Control, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Lactate Dehydrogenase Assay, Staining

Journal: Scientific Reports

Article Title: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

doi: 10.1038/srep17528

Figure Lengend Snippet: Primary rat hepatocytes were transfected with a miR-21 precursor (Pre-miR-21) or control (Pre-miR-C) and treated with 100 μM DCA or no addition for 24 h. ( A ) Real-Time RT-PCR analysis of miR-21 expression (n = 7). ( B ) Immunoblotting of PDCD4 (n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. ( C ) Cell viability (top), cytotoxicity (middle) and caspase-3/7 activity (bottom) measured by the ApoToxGlo TM Triplex assay (n = 5). Results are expressed as mean ± SEM fold change.

Article Snippet: Upon transference onto a nitrocellulose membranes and blocking with 5% milk solution, membranes were probed overnight at 4 °C with primary rabbit polyclonal antibodies against IκB (sc-371), NF-κB p65 (sc-372) and PIDD (sc-67032) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal antibody against active caspase-2 (ab2251; Abcam PLC, Cambridge, United Kingdom), primary goat polyclonal antibody against PDCD4 (sc-27123; Santa Cruz Biotechnology) or primary mouse monoclonal antibodies against β-actin (A5441; Sigma-Aldrich Co.), HDAC1 (05–100; Upstate Biotechonology, Lake Placid, NY, USA), GAPDH (sc-32233; Santa Cruz Biotechnology) and, finally, with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature.

Techniques: Transfection, Control, Quantitative RT-PCR, Expressing, Western Blot, Activity Assay

Journal: Scientific Reports

Article Title: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

doi: 10.1038/srep17528

Figure Lengend Snippet: Primary rat hepatocytes were transfected with a specific siRNA against PDCD4 (siRNA PDCD4) or a control (siRNA C) and treated with 100 μM DCA or no addition for 48 h. ( A ) Immunoblotting of PDCD4 (n = 4). Representative blots are shown. Blots were normalized to endogenous β-actin. ( B ) Cell viability, measured by the ApoTox-Glo TM Triplex assay (n = 4). ( C ) General cell death measured by LDH assay (n = 11). ( D ) Apoptotic cells detected by Hoechst staining. Representative images of control, DCA, PDCD4 silencing and PDCD4 silencing + DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.

Article Snippet: Upon transference onto a nitrocellulose membranes and blocking with 5% milk solution, membranes were probed overnight at 4 °C with primary rabbit polyclonal antibodies against IκB (sc-371), NF-κB p65 (sc-372) and PIDD (sc-67032) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal antibody against active caspase-2 (ab2251; Abcam PLC, Cambridge, United Kingdom), primary goat polyclonal antibody against PDCD4 (sc-27123; Santa Cruz Biotechnology) or primary mouse monoclonal antibodies against β-actin (A5441; Sigma-Aldrich Co.), HDAC1 (05–100; Upstate Biotechonology, Lake Placid, NY, USA), GAPDH (sc-32233; Santa Cruz Biotechnology) and, finally, with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature.

Techniques: Transfection, Control, Western Blot, Lactate Dehydrogenase Assay, Staining

Journal: Scientific Reports

Article Title: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

doi: 10.1038/srep17528

Figure Lengend Snippet: Cells were transfected with a plasmid encoding a constitutively active form of IKK (CA-IKK), NF-κB or an empty vector (pcDNA 3.1) or incubated with 10 μM BAY 11-7085 and treated with 100 μM DCA or no addition as described in Materials and Methods. ( A ) NF-κB transcriptional activity (n = 6). Cells were transfected with a mixture of an inducible NF-κB responsive construct, encoding the firefly luciferase reporter gene, and a constitutively expressing Renilla luciferase construct, as an internal standard control. ( B ) Real-Time RT-PCR analysis of miR-21 (top; n = 5) and immunoblotting of PDCD4 (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. ( C ) Cell viability, measured by the ApoTox-Glo TM Triplex assay (n = 5). ( D ) NF-κB transcriptional activity (n = 4). ( E ) Real-time RT-PCR analysis of miR-21 (top; n = 6) and immunoblotting of PDCD4 (bottom; n = 8). Representative blots are shown. Blots were normalized to endogenous β-actin. ( F ) Cell death, measured by LDH assay (top; n = 8) and caspase-3/7 activity measured by the ApoTox-Glo TM Triplex assay (bottom; n = 6). Results are expressed as mean ± SEM fold change.

Article Snippet: Upon transference onto a nitrocellulose membranes and blocking with 5% milk solution, membranes were probed overnight at 4 °C with primary rabbit polyclonal antibodies against IκB (sc-371), NF-κB p65 (sc-372) and PIDD (sc-67032) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal antibody against active caspase-2 (ab2251; Abcam PLC, Cambridge, United Kingdom), primary goat polyclonal antibody against PDCD4 (sc-27123; Santa Cruz Biotechnology) or primary mouse monoclonal antibodies against β-actin (A5441; Sigma-Aldrich Co.), HDAC1 (05–100; Upstate Biotechonology, Lake Placid, NY, USA), GAPDH (sc-32233; Santa Cruz Biotechnology) and, finally, with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature.

Techniques: Transfection, Plasmid Preparation, Incubation, Activity Assay, Construct, Luciferase, Expressing, Control, Quantitative RT-PCR, Western Blot, Lactate Dehydrogenase Assay

Journal: Scientific Reports

Article Title: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

doi: 10.1038/srep17528

Figure Lengend Snippet: Wistar male rats (n = 6) were treated with 125 mg/kg/day of DCA by oral gavage for 1 and 3 days before sacrifice. Rats in the control group (n = 6) were administered with water as described in Material and Methods. ( A ) Real-Time RT-PCR analysis of miR-21. Results are expressed as mean ± SEM fold change. ( B ) Representative blots and quantitation, in fold-change from controls, of PDCD4, IκB/NF-κB ratio, PIDD-CC and active caspase-2 from liver of rats treated with DCA for 1 day (left) and 3 days (right). Blots were normalized to endogenous β-actin.

Article Snippet: Upon transference onto a nitrocellulose membranes and blocking with 5% milk solution, membranes were probed overnight at 4 °C with primary rabbit polyclonal antibodies against IκB (sc-371), NF-κB p65 (sc-372) and PIDD (sc-67032) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal antibody against active caspase-2 (ab2251; Abcam PLC, Cambridge, United Kingdom), primary goat polyclonal antibody against PDCD4 (sc-27123; Santa Cruz Biotechnology) or primary mouse monoclonal antibodies against β-actin (A5441; Sigma-Aldrich Co.), HDAC1 (05–100; Upstate Biotechonology, Lake Placid, NY, USA), GAPDH (sc-32233; Santa Cruz Biotechnology) and, finally, with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature.

Techniques: Control, Quantitative RT-PCR, Quantitation Assay