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 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: Phospho-proteomics, 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: Antioxidants (Basel, Switzerland)

Article Title: New Treatment for the Cognitive and Emotional Deficits Linked with Paclitaxel-Induced Peripheral Neuropathy in Mice.

doi: 10.3390/antiox11122387

Figure Lengend Snippet: Figure 6. The effects of HRW on the p-ERK 1/2, p-Akt, p-IKBα, and BAX expression in the PFC of PTX-injected animals. The protein levels of p-ERK 1/2/ERK 1/2 (A), p-Akt/Akt (B), p-IKBα/IKBα (D) and BAX (E) in the PFC of PTX-injected mice treated with HRW are shown. Control animals treated with vehicle (VEHI) plus VEHI are also displayed. Representative blots for p-ERK 1/2, ERK 1/2, p-Akt, and Akt (C), p-IKBα, IKBα, BAX and GAPDH (F) are presented. In all graphs, * denotes significant differences with respect to VEHI plus VEHI-treated mice and $ with respect to PTX plus HRW-treated mice (p < 0.05, one-way ANOVA and Student–Newman–Keuls test). Results are represented as mean values ± SEM (n = 3 samples per group).

Article Snippet: Next, the proteins were transferred onto a polyvinylidene fluoride membrane by electrophoresis (120 min) and blocked with phosphate-buffered saline (PBS) + 5% nonfat dry milk, PBS with Tween 20 + 5% bovine serum albumin (BSA), or Tris-buffered saline plus Tween 20 + 5% of nonfat dry milk or BSA, for 75 min. Then, the membranes were incubated with specific rabbit primary antibodies anti-p-ERK 1/2 (1:250), ERK 1/2 (1:250), p-Akt (1:200), Akt (1:200), IKBα (1:200) and BAX (1:250) purchased at Cell Signaling Technology (Danvers, MA, United States); p-IKBα (1:200), 4-HNE (1:150) and HO-1 (1:250) acquired at Abcam (Cambridge, UK); GSTM1 (1:150) and SOD-1 (1:150) from Novus Biologic (Littleton, CO, USA); and NQO1 (1:250) and GAPDH (1:5000) from Merck (Billerica, MA, USA) overnight at 4 ◦C.

Techniques: Expressing, Injection, Control