Journal: PLoS ONE

Article Title: NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

doi: 10.1371/journal.pone.0073409

Figure Lengend Snippet: (A) Dose dependent activation of MyD88 by IL1B in AGS cells. Total RNA was extracted from AGS cells treated with increasing concentration of recombinant IL1B (0, 5, 10ng/ml) for 10 min. Real time PCR analysis for MyD88 was performed in those RNA samples. β-actin was taken as the endogenous control. The graph represents the mean of relative quantification measured from three different experiments +/- SD. (B) Analysis of IL1B induced phosphorylation of MAP3K TAK1. AGS cells were treated with 10ng/ml recombinant IL1B protein for 0, 15 and 30 min and then lysed for western blot analysis with p-TAK1, TAK1 and β–actin antibodies. A representative blot is shown. The band intensities were scanned by imageJ and the normalized mean band intensities of three independent experiments with +/-SD values graphically plotted. (C) Dose dependent activation of NFkB p50 and p65 by TAK1. AGS cells were co-transfected in a dose dependent manner with pCMVTAK1 along with its activator pCMVTAB1 and western blot analysis was done for NFkB p50 and p65 respectively. A representative blot is shown. The band intensities were scanned by imageJ and the normalized mean band intensities of three independent experiments with +/-SD values graphically plotted. (D and E) Effect of TAK1/TAB1on gastrin promoter activity. AGS cells were co-transfected with gastrin luciferase (pGAS-Luc) and either with (D) varying concentrations of pCMVTAK1 along with 0.2 µg of pCMVTAB1 or (E) varying concentrations of pCMVTAB1 along with 0.5 µg of pCMVTAK1.The IL1B (10ng/ml) treatment as control has been included in panel E. Cells were harvested after 48 hr of transfection for luciferase assay. The normalized mean Relative Luciferase Unit/µg protein +/- SD of three different experiments was plotted. (F) Knock down of TAK1 in IL1B-treated AGS cells releases gastrin repression. AGS cells were first transfected with either TAK1 siRNA (80 nM) or control siRNA(80 nM). After twenty four hr these cells were transfected with 0.5 µg of pGAS-Luc.Forty six hr post pGAS-Luc transfection, cells were treated with IL1B (10ng/ml) for two hr and subsequently harvested. Control experiments with only pGAS-Luc transfected and IL1B treated pGAS-Luc transfected AGS cells are also shown. (G) A cartoon showing that IL1B induces NFkB via MyD88/TAK1 to regulate gastrin expression. Stars in panel C indicate statistical significance of the observations.

Article Snippet: IL1B treated AGS cells (2.0×10 6 cells) were subjected to western blot analysis using antibodies against TAK1(1:500 dilution, #4505, Cell Signaling, USA), p-TAK1(1:100 dilution, #4536, Cell Signaling, USA), TAB1(1:500 dilution, #3226, Cell Signaling, USA) NFkB p50 antibody (1:500 dilution, #3035, Cell Signaling, USA), NFkB p65 antibody(1:500 dilution, Sc-7151,Santa Cruz Biotechnology Inc, Santa Cruz, USA and #6956, Cell Signaling, USA), NFkB phosphop65S536 antibody(1:100 dilution, Santa Cruz Biotechnology Inc, Santa Cruz, USA), or Beta -actin (Sigma Aldrich, St. Louis, USA).

Techniques: Activation Assay, Concentration Assay, Recombinant, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Activity Assay, Luciferase, Expressing

Journal: PLoS ONE

Article Title: NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

doi: 10.1371/journal.pone.0073409

Figure Lengend Snippet: (A) Effect of NFkB p65 on gastrin promoter activity. AGS cells were co-transfected with gastrin luciferase (pGAS-Luc) along with 0.2µg of NFkB p65 (pWTp65NFkB). Forty six hr after transfection, cells were either treated or left untreated with 10ng/ml of IL1B for two hr and then harvested for luciferase assay. The normalized mean Relative Luciferase Unit/µg protein +/- SD of three different experiments was plotted. (B and C) Effect of NFkB p65 mutants on gastrin promoter activity. AGS cells were co-transfected with gastrin luciferase (pGAS-Luc) along with (B) either NFkB p65 (pWTp65NFkB) or the lysine NFkB p65 mutants (pK310R p65 NFkB and pK221R p65 NFkB) and (C) with either NFkB p65 (pWTp65NFkB) or with the serine NFkB p65 mutants (pS536A p65 NFkB, pS529A p65 NFkB and pS276A p65 NFkB). Forty six hr after transfection, cells were either treated or left untreated with 10 ng/ml of IL1B for two hr and then harvested for luciferase assay. 0.2µg of wild type or mutant NFkB p65 were used for transfection. The normalized mean Relative Luciferase Unit/µg protein +/- SD of three different experiments was plotted. (D) Analysis of IL1B induced phosphorylation of NFkB p65 at ser536 residue. AGS cells were treated with varying concentrations of recombinant IL1B protein for two hr and then lysed and immunoblotted with anti p-p65NFkB (S536) and anti α-tubulin antibodies. A representative blot is shown. The star in panel C indicates that the repression of transcription of pGAS-Luc with pWTp65NFkB is significantly alleviated when p65S536ANFkB was used both in presence and absence of IL1B.NS indicates that the differences between the repressive activity of pWTp65NFkB and that of the mutant clones on gastrin promoter are not significant.

Article Snippet: IL1B treated AGS cells (2.0×10 6 cells) were subjected to western blot analysis using antibodies against TAK1(1:500 dilution, #4505, Cell Signaling, USA), p-TAK1(1:100 dilution, #4536, Cell Signaling, USA), TAB1(1:500 dilution, #3226, Cell Signaling, USA) NFkB p50 antibody (1:500 dilution, #3035, Cell Signaling, USA), NFkB p65 antibody(1:500 dilution, Sc-7151,Santa Cruz Biotechnology Inc, Santa Cruz, USA and #6956, Cell Signaling, USA), NFkB phosphop65S536 antibody(1:100 dilution, Santa Cruz Biotechnology Inc, Santa Cruz, USA), or Beta -actin (Sigma Aldrich, St. Louis, USA).

Techniques: Activity Assay, Transfection, Luciferase, Mutagenesis, Recombinant, Clone Assay

Journal: PLoS ONE

Article Title: NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

doi: 10.1371/journal.pone.0073409

Figure Lengend Snippet: (A) Effect of HDAC1 on gastrin promoter activity. AGS cells were co-transfected with pGAS-Luc construct and increasing amount of HDAC1 expression vector. Cells were harvested 48 hr after transfection and luciferase activity was measured. The mean luciferase activity of three different experiments +/- SD is graphically plotted. (B and C) Combined effect of NFkB p65 and HDAC1 on gastrin promoter activity. (B) AGS cells were co-transfected with pGAS-Luc construct along with pHDAC1 or pWTp65NFkB or both. Transfected cells were then incubated with either NBD (150µM) for 24 hr or TSA (100nM) for 48 hr or both or left untreated. Cells were harvested after 48 hr of transfection. The mean luciferase activity of three different experiments is represented as fold repression with respect to gastrin promoter alone and graphically plotted. (C) AGS cells were co-transfected with pGAS-Luc in combination with either pWTp65NFkB or pS536A p65 NFkB with and without pHDAC1 expression vector. Cells were harvested after 48 hr of transfection. The mean luciferase activity of three different experiments is represented as Relative Luciferase Unit/µg protein with respect to gastrin promoter alone and graphically plotted. (D) Co-immunoprecipitation assay confirms the association of NFkB p65 with either p300 or HDAC1 in IL1B stimulus specific manner. AGS cells were either treated with 10ng/ml of recombinant IL1B or left untreated for two hr. Lysates were prepared for immunoprecipitation with either p300 or HDAC1. Immunoprecipitated protein complexes were then immunoblotted with NFkB p65 antibody. (E) The band intensities of co-immunoprecipitation experiments were scanned by imageJ and their mean normalized density of three independent experiments with +/-SD values graphically plotted.

Article Snippet: IL1B treated AGS cells (2.0×10 6 cells) were subjected to western blot analysis using antibodies against TAK1(1:500 dilution, #4505, Cell Signaling, USA), p-TAK1(1:100 dilution, #4536, Cell Signaling, USA), TAB1(1:500 dilution, #3226, Cell Signaling, USA) NFkB p50 antibody (1:500 dilution, #3035, Cell Signaling, USA), NFkB p65 antibody(1:500 dilution, Sc-7151,Santa Cruz Biotechnology Inc, Santa Cruz, USA and #6956, Cell Signaling, USA), NFkB phosphop65S536 antibody(1:100 dilution, Santa Cruz Biotechnology Inc, Santa Cruz, USA), or Beta -actin (Sigma Aldrich, St. Louis, USA).

Techniques: Activity Assay, Transfection, Construct, Expressing, Plasmid Preparation, Luciferase, Incubation, Co-Immunoprecipitation Assay, Recombinant, Immunoprecipitation

Journal: PLoS ONE

Article Title: NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

doi: 10.1371/journal.pone.0073409

Figure Lengend Snippet: (A) Direct binding of NFkB heterodimers to gastrin promoter. EMSA was performed with nuclear extracts prepared from IL1B treated (10ng/ml for 2hr.) AGS cells. Supershift assay was performed by incubating the nuclear lysates first with either NFkB p50 or p65 antibody for 30 min and then with radiolabelled gastrin EMSA probe. The chase was performed using corresponding unlabelled oligo probes. NE stands for Nuclear Extract. (B) Gastrin promoter occupancy by NFkB heterodimers and other co-factors. ChIP qPCR analysis was done with anti p50, p65, HDAC1 and p300 antibodies in IL1B (10ng/ml) treated and untreated AGS cells. (C) NFkB associates with HDAC1 and NCoR on the gastrin promoter to bring about histone methylation. ChIP qPCR analysis was done with anti NFkB p65, anti-H3K9Me3 and anti-NCoR in IL1B (10ng/ml) treated and untreated AGS cells.

Article Snippet: IL1B treated AGS cells (2.0×10 6 cells) were subjected to western blot analysis using antibodies against TAK1(1:500 dilution, #4505, Cell Signaling, USA), p-TAK1(1:100 dilution, #4536, Cell Signaling, USA), TAB1(1:500 dilution, #3226, Cell Signaling, USA) NFkB p50 antibody (1:500 dilution, #3035, Cell Signaling, USA), NFkB p65 antibody(1:500 dilution, Sc-7151,Santa Cruz Biotechnology Inc, Santa Cruz, USA and #6956, Cell Signaling, USA), NFkB phosphop65S536 antibody(1:100 dilution, Santa Cruz Biotechnology Inc, Santa Cruz, USA), or Beta -actin (Sigma Aldrich, St. Louis, USA).

Techniques: Binding Assay, Methylation

Journal: CNS Neuroscience & Therapeutics

Article Title: Alpha‐lipoic acid downregulates TRPV1 receptor via NF‐κB and attenuates neuropathic pain in rats with diabetes

doi: 10.1111/cns.13303

Figure Lengend Snippet: ALA inhibited TRPV1 expression via suppressing NF‐κB. A, Western blots for NF‐κB in DRGs innervating the hindpaw from control and STZ‐induced diabetic rats. Bar graph involved mean density relative of β‐actin and NF‐κB from control and STZ‐induced diabetic rats. STZ injection significantly enhanced expression of NF‐κB (N = 4 for each group; * P < .05, compared with CNT, two‐sample t test) in L4‐L6 DRGs. B, Western blots for TRPV1 in L4‐L6 DRGs from diabetic rats treated with p65 siRNA lentivirus and NC siRNA lentivirus, respectively. Graph showed mean density relative to GAPDH for TRPV1. The lentiviruses were intrathecally injected into rats. The expression of TRPV1 significantly reduced after p65 siRNA lentiviruses treatment compared with NC siRNA group (N = 4 for each group, * P < .05, compared with NC siR, two‐sample t test). C, Western blots for NF‐κB in L4‐L6 DRGs from diabetic rats treated with NS and ALA, respectively. Graph showed mean density relative to GAPDH for NF‐κB. ALA treatment strongly reduced the expression of NF‐κB (N = 4 for NS group, N = 3 for ALA group, ** P < .01, compared with NS, two‐sample t test)

Article Snippet: Anti‐TRPV1 (#ACC‐030, Alomone, 1:200), anti‐p65 (#6956, Cell Signaling Technology, 1:1000), and corresponding horseradish peroxidase‐conjugated secondary anti‐rabbit and anti‐mouse antibodies at dilutions of 1:2000 and 1:2000 were used to probe the proteins, respectively.

Techniques: Expressing, Western Blot, Injection

Journal: Pharmaceuticals

Article Title: CK2 Pro-Survival Role in Prostate Cancer Is Mediated via Maintenance and Promotion of Androgen Receptor and NFκB p65 Expression

doi: 10.3390/ph12020089

Figure Lengend Snippet: Effects of increased CK2α expression on AR and NFκB p65 protein levels. ( A ) Immunoblot analysis of RWPE-1 cells stably expressing Flag-CK2α after 45, 62 and 73 days in culture. Control lanes represent either parental RWPE-1 cells or RWPE-1 cells stably expressing empty vector collected simultaneously with Flag-CK2α cells. Proteins detected are indicated on the right side of the blots. Actin signal was used as the loading control. ( B ) Immunoblot analysis of RWPE-1 and C4-2B cell lysates 24 h after transient transfection with Flag-CK2α expression construct. Control lanes represent empty vector transfected cells. β-Tubulin signal was used as the loading control. Arrows indicate correct band.

Article Snippet: CK2β (sc-46666), NFκB p65 P-S529 (sc-101751), AR (sc-818), and Actin (sc-1616) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); NFκB p65 (6956), Cyclin D1 (12231), BiP (3177), IRE1α, CHOP (2895), SQSTM1 p62 (88588), Beta-Tubulin (2128) from Cell Signaling (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Transfection, Construct

Journal: Pharmaceuticals

Article Title: CK2 Pro-Survival Role in Prostate Cancer Is Mediated via Maintenance and Promotion of Androgen Receptor and NFκB p65 Expression

doi: 10.3390/ph12020089

Figure Lengend Snippet: Blocking CK2 expression and activity reduces NFκB p65 protein levels and activation in PCa cells. ( A ) LNCaP cells were transfected with CK2αα’-targeted and control siRNAs. Cells were collected 48 and 72 h post-transfection, as labeled below the blots, for analysis by immunoblot. ( B ) C4-2B and 22Rv1 cells were treated with 20 µM TBB, 40 µM TBB or equivalent concentration of DMSO as indicated. Cells were collected at 24 h for analysis by immunoblot. Vertical lines indicate non-contiguous lanes. For all panels: proteins detected are indicated on the right side of blots and either actin or β-tubulin were used as loading controls. Arrows indicate correct band. Means and 95% confidence intervals for values relative to siControl or DMSO are indicated below each blot.

Article Snippet: CK2β (sc-46666), NFκB p65 P-S529 (sc-101751), AR (sc-818), and Actin (sc-1616) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); NFκB p65 (6956), Cyclin D1 (12231), BiP (3177), IRE1α, CHOP (2895), SQSTM1 p62 (88588), Beta-Tubulin (2128) from Cell Signaling (Danvers, MA, USA).

Techniques: Blocking Assay, Expressing, Activity Assay, Activation Assay, Transfection, Labeling, Western Blot, Concentration Assay

Journal: Pharmaceuticals

Article Title: CK2 Pro-Survival Role in Prostate Cancer Is Mediated via Maintenance and Promotion of Androgen Receptor and NFκB p65 Expression

doi: 10.3390/ph12020089

Figure Lengend Snippet: Anti-CK2 nanocapsule RNAi-based systemic treatment reduces expression of CK2, AR and NFκB p65 in 22Rv1 orthotopic xenograft tumors. ( A ) Orthotopic 22Rv1 tumors were initiated in NOD SCID gamma castrated male mice. When tumors were palpable, mice were treated on days 1, 4 and 7 with TBG-RNAi-CK2 or TBG-RNAi-F7 (control) nanocapsule by tail vein injection (0.02 mg/kg). Tumors were harvested on day 8, 24 h after the last treatment. Tumors were weighed, dissected to remove dead tissue, and reweighed. Mean tumor weights per group are indicated on the left panel and the mean percent of dead tumor tissue removed is indicated on the right panel. TBG-RNAi-CK2, n = 4; TBG-RNAi-F7, n = 3. Error bars indicate standard error. ( B ) Orthotopic xenograft 22Rv1 tumors from mice treated with TBG-RNAi-CK2 or TBG-RNAi-F7 (control) nanocapsule drugs were subjected to immunoblot analysis. Treatments are indicated above the lanes, and proteins detected are indicated to the right of the panels. Mean expression levels and 95% confidence intervals are indicated below the individual blots. Actin was used as a loading control.

Article Snippet: CK2β (sc-46666), NFκB p65 P-S529 (sc-101751), AR (sc-818), and Actin (sc-1616) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); NFκB p65 (6956), Cyclin D1 (12231), BiP (3177), IRE1α, CHOP (2895), SQSTM1 p62 (88588), Beta-Tubulin (2128) from Cell Signaling (Danvers, MA, USA).

Techniques: Expressing, Injection, Western Blot

Journal: Pharmaceuticals

Article Title: CK2 Pro-Survival Role in Prostate Cancer Is Mediated via Maintenance and Promotion of Androgen Receptor and NFκB p65 Expression

doi: 10.3390/ph12020089

Figure Lengend Snippet: Cartoon summary of the influence of CK2 expression and activity on AR and NFκB p65 protein expression and prostate tumor cell survival as described in this work.

Article Snippet: CK2β (sc-46666), NFκB p65 P-S529 (sc-101751), AR (sc-818), and Actin (sc-1616) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); NFκB p65 (6956), Cyclin D1 (12231), BiP (3177), IRE1α, CHOP (2895), SQSTM1 p62 (88588), Beta-Tubulin (2128) from Cell Signaling (Danvers, MA, USA).

Techniques: Expressing, Activity Assay