sirna-iκbα  (Santa Cruz Biotechnology)

Journal: Molecular Vision

Article Title: Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

doi:

Figure Lengend Snippet: Transfection efficiency of IκBα siRNA. To monitor siRNA transfection efficiency, the HCM cells were transfected with Cy3-labled IκBα siRNA using Lipofectamine 2000 reagent, which were viewed at 24 h, 48 h, and 72 h after transfection. Cy3-labled IκBα siRNA (red) was observed in the cytoplasm in HCM cells, and the transfection efficiency was 92% ( A ), 90% ( B ), and 91% ( C ) 24 h, 48 h, and 72 h after IκBα siRNA transfection, respectively.

Article Snippet: The siRNA sequences used for silencing IκBα (GenBank NM_020529 ) were designed, and Cy3-labled IκBα siRNA was synthesized by Guangzhou Ribobio (Guangzhou, China).The sequences of siRNAs were as follows: sense, 5′-CUC CGA GAC UUU CGA GGA A dTdT-3′; antisense, 5′-UUC CUC GAA AGU CUC GG AG dTdT-3′.

Techniques: Transfection

Journal: Molecular Vision

Article Title: Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

doi:

Figure Lengend Snippet: Examination of IκBα mRNA and protein levels in HCM cells 24 h, 48 h, and 72 h after IκBα siRNA transfection. A : IκBα mRNA expression was quantified by real-time RT–PCR. Expression levels were normalized with GAPDH . Error bars represent standard deviations (SD) calculated from three parallel experiments. B : Total cell lysates from HCM cells treated with IκBα siRNA, nonsense control siRNA (NC), and control were analyzed by western blot with IκBα antibody and GAPDH antibody. The arrows indicate IκBα (39 kDa) and GAPDH (36 kDa) bands. The bands were analyzed densitometrically, and the values were normalized with GAPDH, which are represented in the bar graph. The mRNA and protein values of IκBα siRNA, control, and nonsense control siRNA (NC) groups were determined by one-way ANOVA. The double asterisk denotes p<0.01.

Article Snippet: The siRNA sequences used for silencing IκBα (GenBank NM_020529 ) were designed, and Cy3-labled IκBα siRNA was synthesized by Guangzhou Ribobio (Guangzhou, China).The sequences of siRNAs were as follows: sense, 5′-CUC CGA GAC UUU CGA GGA A dTdT-3′; antisense, 5′-UUC CUC GAA AGU CUC GG AG dTdT-3′.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular Vision

Article Title: Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

doi:

Figure Lengend Snippet: Effect of ablation of IκBα on MMP-2 expression and activity 24 h, 48 h, and 72 h after IκBα siRNA transfection. A : MMP-2 mRNA expression in in HCM cells of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control was quantified by real-time RT-PCR. Expression levels were normalized with GAPDH . B : After IκBα siRNA transfection, the conditioned media were collected at the indicated time, concentrated, and analyzed by western blot with MMP-2 antibody. The arrow indicates MMP-2 bands that are analyzed by densitometry and the values were represented in the bar graph. C : The activity of MMP-2 is analyzed by gelatin zymography analysis. The arrows indicate pro-MMP-2 (72-kDa) and active MMP-2 (66-kDa) specific bands. The bands were analyzed by densitometry and are represented in the bar graph. The mRNA, protein, and activity values of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control were determined by one-way ANOVA. An asterisk denotes p<0.05, and a double asterisk indicates p<0.01.

Article Snippet: The siRNA sequences used for silencing IκBα (GenBank NM_020529 ) were designed, and Cy3-labled IκBα siRNA was synthesized by Guangzhou Ribobio (Guangzhou, China).The sequences of siRNAs were as follows: sense, 5′-CUC CGA GAC UUU CGA GGA A dTdT-3′; antisense, 5′-UUC CUC GAA AGU CUC GG AG dTdT-3′.

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

Journal: Molecular Vision

Article Title: Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

doi:

Figure Lengend Snippet: Effect of knockdown IκBα on TIMP-2 and MT1-MMP expression in HCM cells 24 h, 48 h, and 72 h after IκBα siRNA transfection. TIMP-2 ( A ) and MT1-MMP ( C ) mRNA expression in HCM cells of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control was quantified by real-time RT-PCR. Expression levels were normalized with GAPDH . B : After IκBα siRNA transfection, the conditioned media were collected at the indicated time, concentrated, and analyzed by western blot with TIMP-2 antibody. The arrow indicates TIMP-2 (21 kDa). The bands were analyzed by densitometry and represented in the bar graph. D : Total cell lysates from HCM cells transfected with IκBα siRNA , nonsense control siRNA (NC) as well as from the control, respectively, were analyzed by western blot with MT1-MMP antibody. The arrows show MT1-MMP (66 kDa) and internal control, GAPDH (36 kDa). The bands were analyzed by densitometry and represented in the bar graph. The mRNA, protein, and activity values of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control were determined by one-way ANOVA. An asterisk denotes p<0.05, and a double asterisk indicates p<0.01.

Article Snippet: The siRNA sequences used for silencing IκBα (GenBank NM_020529 ) were designed, and Cy3-labled IκBα siRNA was synthesized by Guangzhou Ribobio (Guangzhou, China).The sequences of siRNAs were as follows: sense, 5′-CUC CGA GAC UUU CGA GGA A dTdT-3′; antisense, 5′-UUC CUC GAA AGU CUC GG AG dTdT-3′.

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

Journal: Molecular Vision

Article Title: Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

doi:

Figure Lengend Snippet: Effect of knockdown IκBα on the expression and cellular localization of NF-κBp65 in HCM cells 24 h, 48 h, and 72 h after IκBα siRNA transfection. A : NF-κBp65 mRNA expression in HCM cells of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control was quantified by real-time RT-PCR. Expression levels were normalized with GAPDH . B : Nuclear proteins extracted from IκBα siRNA, nonsense control siRNA (NC), as well as control cells, respectively, were analyzed by western blot with NF-κBp65 antibody. The arrow indicates the NF-κBp65 (75 kDa) band and nucleus internal control, Lamin B (67 kDa). The bands were analyzed densitometrically, and the values were normalized with Lamin B, represented in bar graph. C : Total cell lysates from HCM cells transfected with IκBα siRNA, and nonsense control siRNA (NC) as well as from the control, respectively, were analyzed by western blot with NF-κBp65 antibody. The arrows show the NF-κBp65 (75 kDa) band and the internal control, GAPDH (36 kDa). The mRNA and protein values compared to the control and nonsense control siRNA (NC) were determined by one-way ANOVA. The double asterisk denotes p<0.01. D : The HCM cells were immunostained with NF-κBp65 antibody and analyzed by fluorescence microscopy. A weak nuclear signal of NF-κBp65 (green) was observed in control cells at 24 h, 48 h, and 72 h. After IκBα siRNA transfection, NF-κBp65 translocated from the cytoplasm into the nucleus, and a strong signal of NF-κBp65 was detected in the nucleus at 24 h, 48 h, and 72 h. Cy3 labled IκBα siRNA (red) was observed in the cytoplasm. Cell nuclei were counterstained with DAPI (blue).

Article Snippet: The siRNA sequences used for silencing IκBα (GenBank NM_020529 ) were designed, and Cy3-labled IκBα siRNA was synthesized by Guangzhou Ribobio (Guangzhou, China).The sequences of siRNAs were as follows: sense, 5′-CUC CGA GAC UUU CGA GGA A dTdT-3′; antisense, 5′-UUC CUC GAA AGU CUC GG AG dTdT-3′.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Fluorescence, Microscopy

Journal: Journal of Inflammation (London, England)

Article Title: Different signaling pathways involved in the anti-inflammatory effects of unfractionated heparin on lipopolysaccharide-stimulated human endothelial cells

doi: 10.1186/s12950-020-0238-7

Figure Lengend Snippet: Effects of UFH on nuclear translocation of LPS-stimulated NF-κB p65 by immunofluorescence. Cells were treated with the indicated concentrations of UFH for 15 min, followed by exposure to 10 μg/ml LPS for 1 h. The results are representative of three independent experiments. a Untreated cells. b Control siRNA cells. c IκB-α siRNA cells. d Quantification of the movement of fluorescent label for NF-κB. * P <0.05, compared to the vehicle-treated control group. ** P <0.01, compared to the vehicle-treated control group. # P <0.05, compared to the LPS-treated group. ## P <0.01, compared to the LPS-treated group

Article Snippet: Pre-validated siRNA for human IκB-α (accession number sc-29360) and a negative control (accession number sc-44231) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Translocation Assay, Immunofluorescence, Control