Journal: Molecular cancer therapeutics

Article Title: A Novel Kinase Inhibitor of FADD Phosphorylation Chemosensitizes through the Inhibition of NF-?B

doi: 10.1158/1535-7163.MCT-11-0362

Figure Lengend Snippet: (A) The phosphorylation status of I-κBα was evaluated using AlphaScreen pIκB assay and by (B) western blot analysis in A549 cells following 6-hour treatment with 0, 1, 3, and 6 μM NSC 47147. Results show a decrease in luminescence indicative of pIκB inhibition. Western blot confirms decreasing levels of pIκB proteins and total I-κBα proteins in response to NSC 47147. (C) TNFα induced pIκB levels after 6-hour treatment with and without NSC 47147. A549 cells were stimulated with 10 ng/ml TNFα following pre-treatment with 6 μM NSC 47147. Cellular lysates were subjected to analysis by pIκB AlphaScreen and western blot.

Article Snippet: Phospho-IκB functional assay Phospho-IκB levels were monitored in whole cell lysates using AlphaScreen SureFire p-IκB (S32/36) assay kit, (PerkinElmer, Shelton, CT), a homogenous bead-based assay designed to measure the phosphorylation of endogenous p-IκB (S32/36) in cell lysates, according to the manufacturer’s protocol.

Techniques: Amplified Luminescent Proximity Homogenous Assay, Western Blot, Inhibition

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The inhibitory role of benzo-dioxole-piperamide on the phosphorylation process as an NF-Kappa B silencer.

doi: 10.1016/j.biopha.2021.112471

Figure Lengend Snippet: Fig. 3. Representative images from western blotting analysis and their quantitative analyses of the phospho IKB-α (S32)/GAPDH expression in SVG and CHME3 after treatment for 12 and 24 h; on the left side, there is a comparison graph of the level of phospho-IKB-α (S32) secretion in the cytosol of SVG cells treated with drug molecules along with negative and positive control (A: 12 h treatments and C: 24 h treatments). On the right side, there is the same graph for CHME3 cells treated with drug molecules along with negative and positive control (B: 12 h treatments and D: 24 h treatments). The relevant bolt of each cell line with the best bands was depicted. Nomenclatures used for encoding the blots including 1–2 (1: 12 h treatment of the cells, 2: 24 h treatment of the cells; D5: 2E,4E)-N,5-di(benzo [d][1,3]dioxol-5-yl)penta-2,4-dienamide, Dis: Acetylsalicylic acid (Aspirin/disprin) and LPS: inflamed cells); SVG (astrocyte), CH (CHME3 or microglia). Values are the mean ± S.D. of four samples in one independent experiment. The data were replicated in three repeated independent experiments. The one-way ANOVA followed by post hoc analysis was performed for the comparison of the mean values between D5 treated and the rest of the groups with *p < 0.05; * *p < 0.01 probability, df= 3, F critical= 4.066 for both cell lines at all time points, F values 7979.859, and 14387.24 for SVG and 6026.332, and 13621.58 for CHME3 at 12 and 24 h respectively. The post hoc analysis indicated that D5 significantly reduced the level of phospho-IKB-α (S32) secretion in the cytosol of both cell lines at 12 and 24 h time points compared to Dis treated and LPS-inducing inflamed cells with df= 4, p < 0.001, T critical= 2.776.

Article Snippet: After 30 min, the cells were incubated with monoclonal Rabbit anti-Phospho-IKB alpha (S32) primary antibody (1:4000, Imgenex) in PBS containing 0.1% tween in a humidified chamber overnight at 4 ◦C.

Techniques: Western Blot, Expressing, Comparison, Positive Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The inhibitory role of benzo-dioxole-piperamide on the phosphorylation process as an NF-Kappa B silencer.

doi: 10.1016/j.biopha.2021.112471

Figure Lengend Snippet: Fig. 4. Tracking the NF-kappa B translocation in CHME3 and SVG cell lines through immunocytochemistry using anti-phospho-IKB alpha (S32) antibody for 24 h; Representative images of hematoxylin/DAB immunocytochemistry were shown at 400-fold magnification to demonstrate effects of D5 in comparison with Dis on LPS-induced decomposition of NF-kB complex in the cytosol and subcellular localization of phospho-IkB-α S32. DAB (Brown)-labeled HRP secondary antibody against anti-phospho-IKB alpha (S32) rabbit monoclonal antibody was shown in the cytoplasms peripheral to nuclei counterstained with Hematoxylin (dark blue to black) in control astrocyte (SVG) and microglia (CHME3) group (D, H). In the LPS-treated astrocyte and microglia group, Phospho-IKB alpha (S32) subunit im munoreactivities were shown in the cytosol (C, G). D5 and Dis treatments markedly protect the NF-kB complex in the inactive form in the cytosol of astrocytic glial and microglial cells by reducing the level of cytoplasmic radical Phospho-IKB alpha (S32) ((B, F) respectively. Scale bar: 50 µm.

Article Snippet: After 30 min, the cells were incubated with monoclonal Rabbit anti-Phospho-IKB alpha (S32) primary antibody (1:4000, Imgenex) in PBS containing 0.1% tween in a humidified chamber overnight at 4 ◦C.

Techniques: Translocation Assay, Immunocytochemistry, Comparison, Labeling, Control

Journal: PLoS ONE

Article Title: Inhibition of IGF-1 Signalling Enhances the Apoptotic Effect of AS602868, an IKK2 Inhibitor, in Multiple Myeloma Cell Lines

doi: 10.1371/journal.pone.0022641

Figure Lengend Snippet: 5.A AS602868 and anti-IGF-1R antibody decreased the level of NF-κB proteins pathways. RPMI8226 cells were treated for 16 h with 10 µg/mL anti-IGF-1R antibody or/and 10 µM AS602868 at 37°C. 5.B AS602868 and anti-IGF-1R antibody inhibited the expression of NF-κB regulated genes in multiple myeloma (IKB, IGF1, IL-6 and ICAM-1). Mean± SD of three experiments ** P <0.001: * P <0.05 versus control.

Article Snippet: The antibodies used were directed against Bcl-2 (M0887, 1/500, Dako, Denmark), Bcl-XL (sc-634,1/500, Santa cruz, USA), IGF-1 (sc-1422, 1/500, Santa cruz, USA), P21(sc-397, 1/500, Santa cruz, USA), P53 (clone DO7, 1/1000, Dako, Denmark), IkBα(ser32/ser36, IMG-156,1/500 IMGENEX, USA), Bim (2819), Bad (610392, 1/250, BD transduction, USA), IKKβ (2684), IKKβ (2681), phospho-IKBα (S32/S36) (9426), NF-κB p65 (C22B4), phospho-NF-κB p65 (Ser536), 4EBP 1(9644), Phospho 4EBP-1(2855), p70S6k (2708), phospho p70S6k (9209), cyclin E (4129), cyclin A (4656) (1/1000 or 1/2000, Cell Signalling, USA), Akt (IMG-5411-1, 1/250, Imgenex, USA), phospho Akt (IMG-187-A, 1/500, Imgenex, USA)).

Techniques: Expressing