Journal: The Journal of Neuroscience

Article Title: The Ubiquitin E3 Ligase TRAF6 Exacerbates Ischemic Stroke by Ubiquitinating and Activating Rac1

doi: 10.1523/JNEUROSCI.1751-17.2017

Figure Lengend Snippet: Antibodies used for immunoblot analyses

Article Snippet: The protein bands were detected using a Bio-Rad imaging system and specific protein expression levels were normalized to GAPDH protein. table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Antibody Manufacturer Catalog # Sources of species RRID TRAF6 Abcam Ab40675 Rabbit AB_778573 Bax CST 2772 Rabbit AB_329921 Bcl-2(50E 3 ) CST 2870 Rabbit AB_2290370 Cleaved Caspase3(Asp175) CST 9661 Rabbit AB_2341188 Caspase-3 CST 9662 Rabbit AB_331439 p-IkBa(ser32/36) CST 9246L Mouse AB_2267145 IkBa(L35A5) CST 4814 Mouse AB_390781 P-Ikkβ(Y199) Abcam Ab59195 Rabbit AB_943810 IKKβ CST 8943S Rabbit AB_11024092 P-NF-kB p65(Ser536) CST 3033 Rabbit AB_331284 p65 CST 4764 Rabbit AB_823578 P47-phox(P366) Bioworld BS3261 Rabbit AB_1662897 NOXA2/P67 phox Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab109366","term_id":"37572651","term_text":"AB109366"}} Ab109366 Rabbit AB_10860640 SD1(SOD1) Abcam Ab13498 Rabbit AB_300402 Heme Oxygenase 1(HO-1) Abcam Ab13243 Rabbit AB_299790 p-Rac1/cdc42(Ser71) CST 2461S Rabbit AB_2300703 Rac1/2/3 CST 2467 Rabbit AB_2176150 HA Sigma-Aldrich H6908 Rabbit AB_260070 Flag Sigma-Aldrich F3165 Mouse AB_259529 Myc MBL M192-3 Mouse AB_11160947 Ub CST 3933S Rabbit AB_2180538 His CST 12698 Rabbit Gst CST 2624S Mouse AB_10692101 GAPDH(14C10) CST 2118 Rabbit AB_561053 Open in a separate window Antibodies used for immunoblot analyses Cell culture and in vitro manipulation.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Beta Defensin-2 Is Reduced in Central but Not in Distal Airways of Smoker COPD Patients

doi: 10.1371/journal.pone.0033601

Figure Lengend Snippet: 16-HBE cells were cultured in the presence and in the absence of IL-1 β and of CSE (10%) (n = 3) (see for details). A ) Expression of HBD2 m-RNA in 16-HBE by real time PCR. GAPDH gene expression was used as endogenous control for normalization. Relative quantitation of mRNA was carried out with comparative CT method. (mean±SD). * p<0.05 versus baseline. ** p<0.05 versus IL-1 beta. B ) Representative experiment (one out of three experiments) showing the expression of HBD2 protein in 16-HBE by flow cytometry. The expression of HBD2 is expressed as percentage of HBD2 positive cells. C ) Evaluation of p-IkBa or t-IkBa by western blot analysis. Membranes were then stripped and incubated with goat polyclonal anti–ß-actin. Representative western blot analysis (one out of three experiments). Lane1 = baseline; lane 2 = CSE 10%;lane 3 = IL1 beta; lane 4 = CSE+IL1 beta. D ) ChiP assay using anti-NFkB antibody and PCR using primers (forward 5′-GGTGTGAATGGAAGGAACTCA-3′ reverse 5′-TTCAGCTCCTGGGGATGATAC-3′ ) spanning the promoter region of HBD2 gene were performed (see for details) One out of two experiments is shown. Lane 1 = DNA marker; Lane 2 = baseline; lane 3 = CSE 10%; lane 4 = IL1 β; lane 5 = CSE+IL1 β.

Article Snippet: All blots were first probed using a rabbit polyclonal antibody anti-pIkBa (1∶500) (Cell Signaling Technology Inc) and a rabbit polyclonal antibody anti-IkBa (1∶1000) (Cell Signaling Technology Inc).

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Control, Quantitation Assay, Flow Cytometry, Western Blot, Incubation, Marker

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: Journal of the Formosan Medical Association = Taiwan yi zhi

Article Title: Molecular signaling and mechanisms of low-level laser-induced gene expression in cells involved in orthodontic tooth movement.

doi: 10.1016/j.jfma.2023.09.011

Figure Lengend Snippet: Figure 6 Graph and Western blot images showing the response of p-IkBa and IkBa levels to 10-J/cm2 LLLT in each cell type after stimulation with different timings. Certain group was treated with NF-kB inhibitor (12.5 mM BAY 11e7082) 1hr before LLLT. (A) Graph demonstrating the fold change of p-IkBa to IkBa. *p < 0.05 versus control group, **p < 0.01 versus control group. Images of Western blot results for iPDL (B), MG63 (C), and M1 macrophage-like cells (D).

Article Snippet: Primary antibodies: p- IkBa mouse monoclonal antibody (1:200; sc-8404, Santa Cruz Biotechnology Inc., Heidelberg, Germany), IkBamouse monoclonal antibody (1:200; sc-1634, Santa Cruz Biotechnology Inc., Heidelberg, Germany), and b-actin rabbit polyclonal antibody (1:5000; ab8227, Abcam, Cambridge, UK).

Techniques: Western Blot, Control