nf κb responsive promoter  (Addgene inc)

Journal: Current Research in Toxicology

Article Title: Wheatgrass inhibits the lipopolysaccharide-stimulated inflammatory effect in RAW 264.7 macrophages

doi: 10.1016/j.crtox.2021.02.005

Figure Lengend Snippet: Effect of wheatgrass extract on the transactivation of NF-κB promoter in LPS-stimulated RAW 264.7 cells. Raw 264.7 cells were transfected with pNFκB-Luc and β-gal plasmids and then exposed to 1 µg/ml of LPS in the presence or absence of W5 (A) and W7 (B) for 12 h followed by estimation of luciferase activities. Results are mean ± SEM of three independent experiments. * and # indicate p < 0.05 as compared to vehicle-treated control and LPS-stimulated cells, respectively. W5, 5-day old wheatgrass methanol extract; W7, 7-day old wheatgrass methanol extract; LPS, lipopolysaccharide.

Article Snippet: For this assay, RAW 264.7 cells were co-transfected with a p-NF-κB-Luc reporter, containing mammalian NF-κB response element in its promoter region, and β-gal plasmids (both procured from Promega Corporation, Madison, WI, USA), where the latter was used as an internal control.

Techniques: Transfection, Luciferase

Journal: Journal of neurochemistry

Article Title: Palmitate induced C/EBP homologous protein activation leads to NF-κB -mediated increase in BACE1 activity and Amyloid beta genesis

doi: 10.1111/jnc.14292

Figure Lengend Snippet: List of monoclonal and polyclonal antibodies used in the study

Article Snippet: The luciferase reporter constructs encoding the NF-κB response elements in the BACE1 promoter region were purchased from SwitchGear Genomics (Active Motif, La Jolla, CA).

Techniques:

Journal: Journal of neurochemistry

Article Title: Palmitate induced C/EBP homologous protein activation leads to NF-κB -mediated increase in BACE1 activity and Amyloid beta genesis

doi: 10.1111/jnc.14292

Figure Lengend Snippet: List of primers used for ChIP analysis

Article Snippet: The luciferase reporter constructs encoding the NF-κB response elements in the BACE1 promoter region were purchased from SwitchGear Genomics (Active Motif, La Jolla, CA).

Techniques: Sequencing

Journal: Journal of neurochemistry

Article Title: Palmitate induced C/EBP homologous protein activation leads to NF-κB -mediated increase in BACE1 activity and Amyloid beta genesis

doi: 10.1111/jnc.14292

Figure Lengend Snippet: (A) Representative western blots show that inhibition of NF-κB activation, either by pretreatment (for 3 hours) of the cells with sc-514, a IKKβ-specific inhibitor, or by knocking-down IKKβ expression using shRNA, or by ectopic overexpression of IκBα, significantly attenuates the exogenous palmitate treatment (100μM for 24 hours) - induced increase in BACE1 protein levels accompanied by a decrease in the amyloidogenic processing of AβPP as evidenced by a decrease in the palmitate-induced increase in sAPPβ and CTFβ levels concomitant with an increase in the palmitate-induced decrease in sAPPα and CTFα levels in the whole cell homogenates from SH-SY5Y-APPSwe cells. (B) Inhibition of NF-κB activation attenuates the palmitate-induced increase in BACE1 mRNA expression and BACE1 activity in SH-SY5Y-APPSwe cells. (C) ChIP analysis (Inset I) and Dual luciferase reporter assay (Inset II) show that exogenous palmitate treatment increases the binding of the p65 subunit of NF-κB to the BACE1 promoter region and the NF-κB transcriptional activity, respectively, in SH-SY5Y-APPSwe cells. (D) Dual luciferase reporter assays show that exogenous palmitate treatment increases the transactivation of the BACE1 promoter that is contingent on NF-κB transcriptional activity. Data is expressed as Mean ± S.D and includes determination made in four (n=4) separate cell culture experiments. *p < 0.05, ***p < 0.001 versus BSA-treated control cells or BSA-treated GFP knock-down cells or BSA-treated pCMV-EV transfected cells; ††p < 0.01, versus palmitate-treated control cells or palmitate-treated GFP knock-down cells or palmitate-treated pCMV-EV transfected cells.

Article Snippet: The luciferase reporter constructs encoding the NF-κB response elements in the BACE1 promoter region were purchased from SwitchGear Genomics (Active Motif, La Jolla, CA).

Techniques: Western Blot, Inhibition, Activation Assay, Expressing, shRNA, Over Expression, Activity Assay, Luciferase, Reporter Assay, Binding Assay, Cell Culture, Transfection

Journal: Journal of neurochemistry

Article Title: Palmitate induced C/EBP homologous protein activation leads to NF-κB -mediated increase in BACE1 activity and Amyloid beta genesis

doi: 10.1111/jnc.14292

Figure Lengend Snippet: (A, B) Representative western blots show that knocking-down CHOP expression using a RNAi approach significantly attenuates the palmitate-induced increase in NF-κB activation as evidenced by a pronounced decrease in the palmitate-induced increase in phosphorylation of IKKβ and IκBα (A) followed by a significant reduction in the palmitate-induced increase in the nuclear translocation of the p65 and p50 subunits of NF-κB (B) in SH-SY5Y-APPSwe cells. (C) ChIP analysis shows that knocking-down CHOP expression attenuates the palmitate-induced increase in the p65 NF-κB binding to the proximal BACE1 promoter in SH-SY5Y-APPSwe cells. (D) Luciferase reporter assays demonstrate that knocking-down CHOP expression significantly mitigates the exogenous palmitate treatment-induced increase in NF-κB driven BACE1 promoter transactivation in SH-SY5Y-APPSwe cells. Data is expressed as Mean ± S.D and includes determination made in four (n=4) separate cell culture experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus BSA-treated GFP knock-down cells or BSA-treated scrambled siRNA transfected cells; ††p < 0.01, versus palmitate-treated GFP knock-down cells or palmitate-treated scrambled siRNA transfected cells.

Article Snippet: The luciferase reporter constructs encoding the NF-κB response elements in the BACE1 promoter region were purchased from SwitchGear Genomics (Active Motif, La Jolla, CA).

Techniques: Western Blot, Expressing, Activation Assay, Translocation Assay, Binding Assay, Luciferase, Cell Culture, Transfection

Journal: Journal of neurochemistry

Article Title: Palmitate induced C/EBP homologous protein activation leads to NF-κB -mediated increase in BACE1 activity and Amyloid beta genesis

doi: 10.1111/jnc.14292

Figure Lengend Snippet: (A, B) Representative western blots show that Chop−/− mice fed a palmitate-enriched diet do not exhibit, to the same degree, the increase in NF-κB activation as evidenced by the lack of a pronounced increase in the phosphorylation of IKKβ and IκBα thereby precluding the subsequent increase in the nuclear translocation of the p65 and p50 subunits of NF-κB, in the cortex (A) and the hippocampus (B), compared to the C57BL/6J wild-type mice fed a palmitate-enriched diet. (C, D) ChIP analysis shows that Chop−/− mice fed a palmitate-enriched diet have significantly lower levels of p65 NF-κB bound to the proximal BACE1 promoter in the cortex (C) and the hippocampus (D), compared to the C57BL/6J wild-type mice fed a palmitate-enriched diet. Data is expressed as Mean ± S.D and includes determination made in six (n=6) different animals from each group. ***p < 0.001, versus C57BL/6J wild-type mice fed a control chow diet; ††p < 0.01, versus C57BL/6J wild-type mice fed a palmitate-enriched diet; △p < 0.05, △△p < 0.01 versus Chop−/− mice fed a control chow diet.

Article Snippet: The luciferase reporter constructs encoding the NF-κB response elements in the BACE1 promoter region were purchased from SwitchGear Genomics (Active Motif, La Jolla, CA).

Techniques: Western Blot, Activation Assay, Translocation Assay

Journal: Journal of neurochemistry

Article Title: Palmitate induced C/EBP homologous protein activation leads to NF-κB -mediated increase in BACE1 activity and Amyloid beta genesis

doi: 10.1111/jnc.14292

Figure Lengend Snippet: (A, B) Representative western blots show that CHOP ectopic overexpression-induced increase in BACE1 protein levels and amyloidogenic processing of AβPP is abrogated in cells with compromised NF-κB activation achieved as a result of knocking-down IKKβ expression or ectopic overexpression of IκBα, in SH-SY5Y-APPSwe cells. (C, D) Inhibition of NF-κB activation, as a result of knocking-down IKKβ expression or ectopic overexpression of IκBα, also abrogates the CHOP ectopic overexpression-induced increase in BACE1 mRNA expression (C) and BACE1 activity (D) in SH-SY5Y-APPSwe cells. Data is expressed as Mean ± S.D and includes determination made in four (n=4) separate cell culture experiments. △△p < 0.01, versus BSA-treated GFP shRNA transfected CHOP overexpressing cells; ●●●p<0.001 versus palmitate-treated GFP shRNA transfected CHOP overexpressing cells; ◆◆p<0.01, ◆◆◆p<0.001 versus BSA-treated pCMV-EV transfected CHOP overexpressing cells; ■■p<0.01, versus palmitate-treated pCMV-EV transfected CHOP overexpressing cells.

Article Snippet: The luciferase reporter constructs encoding the NF-κB response elements in the BACE1 promoter region were purchased from SwitchGear Genomics (Active Motif, La Jolla, CA).

Techniques: Western Blot, Over Expression, Activation Assay, Expressing, Inhibition, Activity Assay, Cell Culture, shRNA, Transfection

Journal: Journal of neurochemistry

Article Title: Palmitate induced C/EBP homologous protein activation leads to NF-κB -mediated increase in BACE1 activity and Amyloid beta genesis

doi: 10.1111/jnc.14292

Figure Lengend Snippet: (A, B) Representative western blots show that the CHOP knock down-induced mitigation in BACE1 protein levels and amyloidogenic processing of AβPP is completely lost in cells with constitutive NF-κB activation achieved as a result of knocking-down IκBα expression or by ectopic overexpression of constitutive dominant active IKKβ mutant (IKKβ S177E), in SH-SY5Y-APPSwe cells. (C, D) Constitutive activation of NF-κB, as a result of knocking-down IκBα expression or ectopic overexpression of constitutive dominant active IKKβ mutant (IKKβ S177E), also completely reverses the CHOP knock down-induced decrease in BACE1 mRNA expression (C) and BACE1 activity (D) in SH-SY5Y-APPSwe cells. (E, F) ELISA immunoassays show that constitutive NF-κB activation, completely reverses the CHOP knocked-down - induced decrease in the levels of the intracellular Aβ1-42 species in the whole cell lysates (E) and secreted Aβ1-42 species in the conditioned media (F), from SH-SY5Y-APPSwe cells. Data is expressed as Mean ± S.D and includes determination made in four (n=4) separate cell culture experiments. △△p < 0.01, versus BSA-treated pCMV2-EV transfected CHOP knocked-down cells; ●●●p<0.001, versus palmitate-treated pCMV2-EV transfected CHOP knocked-down cells; ◆◆p<0.01, versus BSA-treated GFP shRNA transfected CHOP knocked-down cells; ■■■p<0.001, versus palmitate-treated GFP shRNA transfected CHOP knocked-down cells.

Article Snippet: The luciferase reporter constructs encoding the NF-κB response elements in the BACE1 promoter region were purchased from SwitchGear Genomics (Active Motif, La Jolla, CA).

Techniques: Western Blot, Activation Assay, Expressing, Over Expression, Mutagenesis, Activity Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Transfection, shRNA