Journal: Science Advances

Article Title: Breast tumors interfere with endothelial TRAIL at the premetastatic niche to promote cancer cell seeding

doi: 10.1126/sciadv.add5028

Figure Lengend Snippet: ( A ) Protein levels of p38 (total and ph-Thr 180 /Tyr 182 ) and IκBα (total and ph-Ser 32/36 ) in shSCR or shTRAIL HUVECs. For each time point, the densitometry fold change of shTRAIL toward shSCR is indicated. For TRAIL knockdown efficiency, see Fig. 6B . ( B ) Immunofluorescence staining of NF-κB (green) and Hoechst (blue) in shSCR or shTRAIL HUVECs. Scale bars, 20 μm. ( C and D ) GSEA enrichment plots showing the comparison of the gene expression profiles in lung ECs sorted from tumor-free induced EC W/W and EC ΔT10 mice. ( E ) Coimmunoprecipitation (co-IP) of DR4, DR5, or IgG control in HUVECs at day 3, showing protein levels of IKKα, ΙΚΚβ, caspase-8, RIP1, NEMO, FADD, DR4, DR5, and TRAIL. Input = WCE. ( F ) Protein levels of p38 (total and ph-Thr 180 /Tyr 182 ) and total IκBα in shSCR and shTRAIL HUVECs at day 5 treated with the pan-caspase inhibitor qVD (50 μM) since day 0. For TRAIL knockdown efficiency and caspase cleavage inhibition, see Fig. 6F . ( G to J ) mRNA expression of ICAM1 (G and I) and SELE (H and J) in shSCR and shTRAIL HUVECs at day 6 treated either with the IκB inhibitor IKK-16 (NF-κBi; 2,5 μM) (G and H) or with the p38 inhibitor SB203580 (p38i; 10 μM) (I and J) since day 4. ( K and L ) Protein levels of ICAM1, E-Selectin, and TRAIL in shSCR and shTRAIL HUVECs at day 6 treated either with NF-κBi (2.5 or 5 μM) (K) or with p38i (1 or 10 μM) (L) since day 4. ( M and N ) E0771 cancer cells adhered to shSCR and shTRAIL HUVEC monolayers at day 6 after 1 hour of contact. HUVECs were treated either with NF-κBi (2.5 μM) for 24 hours (M) or with p38i (10 μM) for 48 hours (N). All graphs show means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Subsequently, membranes were incubated overnight at 4°C with the following primary antibodies diluted in PBST containing 5% BSA or nonfat dry milk (according to the manufacturer’s recommendations): human TRAIL (Cell Signaling Technology, 3219; 1:1000), murine TRAIL (R&D, AF1121; 1:500), murine DcR2 (R&D, MAB1816; 1:500), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, 2218; 1:1000), VE-cadherin (BD Biosciences, 610252; 1:1000), tubulin–horseradish peroxidase (HRP) (Abcam, ab21058; 1:2000), pro–caspase-3 (Cell Signaling Technology, 9668; 1:1000), cleaved caspase-3 (Cell Signaling Technology, 9116; 1:1000), pro–caspase-8 (Cell Signaling Technology, 4790 or 9746; 1:1000), cleaved caspase-8 (Cell Signaling Technology, 9496; 1:1000), vinculin (Sigma-Aldrich, V9131; 1:2000), ICAM1 (Cell Signaling Technology, 67836; 1:1000), E-Selectin (Santa Cruz Biotechnology, sc-137054; 1:800), p38 ph-Thr 180 /Tyr 182 (Cell Signaling Technology, 4511; 1:1000), p38 (Cell Signaling Technology, 8690; 1:1000), IκBα ph-Ser 32/36 (Cell Signaling Technology, 9246; 1:1000), IκBα (Cell Signaling Technology, 4814; 1:1000), DR4 (Cell Signaling Technology, 42533; 1:1000), DR5 (Cell Signaling Technology, 8074; 1:1000), IKKα (Cell Signaling Technology, 2682; 1:1000), IKKβ (Cell Signaling Technology, 8943; 1:1000), RIP1 (Cell Signaling Technology, 3493; 1:1000), NEMO (BD Biosciences, 611306; 1:1000), and FADD (Cell Signaling Technology, 2782; 1:1000).

Techniques: Immunofluorescence, Staining, Expressing, Co-Immunoprecipitation Assay, Inhibition

Journal: Arthritis Research & Therapy

Article Title: Flow cytometric assessment of the signaling status of human B lymphocytes from normal and autoimmune individuals

doi: 10.1186/ar1155

Figure Lengend Snippet: Assessment of extracellular regulated kinase (ERK), jun N-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK) activity in human B lymphocytes by intracellular flow cytometric analysis. R2G6 cells (3 × 10 5 ) were fixed, permeabilized, incubated in the presence or absence of a blocking peptide and stained with isotype matched control antibodies or phosphospecific antibodies that recognize pERK, pJNK and p-p38 (a) immediately or (b) following incubation with increasing amounts of rCD154 for 15 min at 37°C. Dot plots of cell size on the x-axis versus fluorescence intensity in the channel that detects the fluorochrome conjugated secondary antibody on the y-axis are shown in (a). Histograms of fluorescence intensity of the channel that detects the fluorochrome conjugated secondary antibody are shown in (a) and (b). The percentage of positive cells and the mean fluorescence intensity (MFI) of positive staining are indicated. The solid line indicates the division between negative background staining of isotype-matched control antibody and positive staining with the pMAPK antibody. (c) Western blot analysis of pERK, pJNK or p-p38 expression in R2G6 cells following incubation with rCD154 for 15 min at 37°C.

Article Snippet: Cells were analyzed for phosphorylated and degraded IκB proteins by staining for 30 min at 4°C with 3 μg mouse IgG1 anti-IκBα mAb (Becton Dickinson/Transduction Laboratories), phosphospecific IκBα mAb (Ser 32/ser 36; Cell Signaling Technologies) or the isotype-matched control MOPC (ATCC).

Techniques: Activity Assay, Incubation, Blocking Assay, Staining, Fluorescence, Western Blot, Expressing

Journal: Arthritis Research & Therapy

Article Title: Flow cytometric assessment of the signaling status of human B lymphocytes from normal and autoimmune individuals

doi: 10.1186/ar1155

Figure Lengend Snippet: Multiparameter intracellular flow cytometry reveals elevated percentages of B lymphocytes in the periphery of systemic lupus erythematosus (SLE) patients with spontaneous activation of extracellular regulated kinase (ERK), jun N-terminal kinase (JNK) and p38 mitogen activated protein kinases (MAPKs). Peripheral B lymphocytes isolated from SLE patients ( n = 7) or nonautoimmune normal individuals ( n = 7) were fixed, permeabilized and stained with phosphospecific antibody for pIκBα, pERK, pJNK or p-p38. The mean ± SEM percentages of CD19 + B cells positive for each phospho-Ab are shown graphically. * P < 0.05 by Student's t test.

Article Snippet: Cells were analyzed for phosphorylated and degraded IκB proteins by staining for 30 min at 4°C with 3 μg mouse IgG1 anti-IκBα mAb (Becton Dickinson/Transduction Laboratories), phosphospecific IκBα mAb (Ser 32/ser 36; Cell Signaling Technologies) or the isotype-matched control MOPC (ATCC).

Techniques: Flow Cytometry, Activation Assay, Isolation, Staining

Journal: PLoS ONE

Article Title: Modulatory Effects of Vasoactive Intestinal Peptide on Intestinal Mucosal Immunity and Microbial Community of Weaned Piglets Challenged by an Enterotoxigenic Escherichia coli (K88)

doi: 10.1371/journal.pone.0104183

Figure Lengend Snippet: Cytosolic occludin, IκB-α, p-IκB-α, p44/p42 (ERK1/2), p-p44/p42, p38, p-p38, JNK/SAPK, p-JNK/SAPK, p65, TLR4, TLR2, MyD88, nuclear NF-κB p65 proteins and actin were detected by western blot analysis. Each results is the mean (n = 6)±S.E.M. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Mainly Antibodies used in the experiment were as following: IκB-α (EPI), Ser 32/36 -phosphorylated IκB-α (CST), NF-κB p65 (Santa), p44/42 MAPK (ERK1/2) (Bioworld), p-p44/42 (p-ERK1/2) (Bioworld), JNK/SAPK (Santa), p-JNK/SAPK(Santa), p38 MAPK (EPI), p-p38 MAPK(CST), TLR2(EPI), TLR4(Santa), MyD88(Abcam), occludin and β - actin (Santa).

Techniques: Western Blot