signalsilence creb sirna ii  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Notch signaling facilitates hepatitis B virus covalently closed circular DNA transcription via cAMP response element-binding protein with E3 ubiquitin ligase-modulation

doi: 10.1038/s41598-018-38139-5

Figure Lengend Snippet: Notch signaling affects cAMP response element-binding protein (CREB) 1 and CREB-binding protein (CBP) gene expression. ( A ) RTD-PCR analysis of CREB1 and CBP gene levels after Notch inhibition by Jagged1 shRNA transfection. ( B ) Protein expression levels were confirmed by western blotting using anti-CREB, anti-phospho-CREB (Ser133), and anti-CBP antibodies after Jagged1 shRNA transfection. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (Control-shRNA = 1). ( C ) CREB1 and CBP gene expression levels after DAPT treatment. ( D ) Protein expression of CREB, phospho-CREB (Ser133), and CBP after DAPT treatment. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (DMSO = 1). ( E ) RTD-PCR analysis of CREB1 and CBP gene expression levels after MG-132 treatment. ( F ) CREB, phospho-CREB (Ser133), and CBP protein expression by western blotting after MG-132 treatment. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (DMSO = 1). Quantitative gene expression data represent the mean ± standard deviation of three independent experiments and were normalized to the expression levels of human GAPDH. * P < 0.05 and ** P < 0.01 versus the corresponding control shRNA and DMSO (Control-shRNA, DMSO = 1).

Article Snippet: Gene-specific endoribonuclease-prepared small interfering RNA (siRNA; Sigma) for human ITCH and NUMB and SignalSilence ® CREB-siRNA II (Cell Signaling Technology ) were used to silence gene expression.

Techniques: Binding Assay, Expressing, Inhibition, shRNA, Transfection, Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: Notch signaling facilitates hepatitis B virus covalently closed circular DNA transcription via cAMP response element-binding protein with E3 ubiquitin ligase-modulation

doi: 10.1038/s41598-018-38139-5

Figure Lengend Snippet: The CREB (pSer133CREB)-CBP cascade mediates HBV replication. RTD-PCR analysis of CBP gene expression after 10 µM Fsk ( A ) and 5 µM H-89 ( B ) treatment for 120 h. ( C ) Western blot analysis for CBP and phospho-CREB (Ser133) protein expression after 10 µM Fsk and 5 µM H-89 treatment for 120 h. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (DMSO = 1). The HBV cccDNA level was increased in the presence of 10 µM Fsk (120 h), as determined by RTD-PCR ( D ) and Southern blot analysis ( E ). The HBV cccDNA level was decreased in the presence of 5 µM H-89 (120 h), as determined by RTD-PCR ( F ) and Southern blot analysis ( G ). ( H ) CREB mRNA and protein levels detected by RTD-PCR and western blotting were decreased after transfection with CREB-siRNA. ( I ) RTD-PCR quantification of cccDNA accumulation in HepG2.2.15.7 and HepAD38 cells transfected with CREB-siRNA or control-siRNA. ( J ) Southern blot analysis confirming lower cccDNA levels in HepG2.2.15.7 and HepAD38 cells transfected with CREB-siRNA compared with control-siRNA. Results are expressed as the number of cccDNA copies per genomic μg (mean ± standard deviation) from three independent experiments. * P < 0.05 and ** P < 0.01 versus the corresponding DMSO. Quantitative gene expression data represent the mean ± standard deviation of three independent experiments and were normalized to the expression levels of human GAPDH. * P < 0.05 and ** P < 0.01 versus Control-siRNA (Control-siRNA = 1). Abbreviations: DM, DMSO; siCON, Control-siRNA; siCREB, CREB-siRNA.

Article Snippet: Gene-specific endoribonuclease-prepared small interfering RNA (siRNA; Sigma) for human ITCH and NUMB and SignalSilence ® CREB-siRNA II (Cell Signaling Technology ) were used to silence gene expression.

Techniques: Expressing, Western Blot, Southern Blot, Transfection, Standard Deviation