Journal: International immunology

Article Title: Increased urinary exosomal SYT17 levels in chronic active antibody-mediated rejection after kidney transplantation via the IL-6 amplifier.

doi: 10.1093/intimm/dxaa032

Figure Lengend Snippet: Fig. 2. NFκB and STAT3 activation in tubular cells of CAAMR patients. (A) Immunohistochemistry of phosphorylated NFκB p65 (p-p65) and phosphorylated STAT3 (p-STAT3) using biopsy specimens from KTx patients with NED, IF/TA, CNI-T or CAAMR. Representative images of a single patient sample are shown for each group. Scale bars are 100 μm. Red arrows are examples of p-p65 and p-STAT3 double-positive cells. (B) Immunohistochemistry of co-staining with p-p65 (brown) and SYT17 (red), or p-STAT3 (brown) and SYT17 (red) in the tubular and glomeruli regions of specimens from a CAAMR patient. Examples of SYT17-expressing cells with phosphorylated p65 and STAT3 are indicated with red arrows. Representative images are shown. Scale bars are 100 μm.

Article Snippet: For double staining of SYT17 with STAT3 pY705 or with phospho NFκB p65 Ser276, the Polink DS-RR-Hu/Ms A Kit (GBI Labs, Bothell, WA, USA) was used with a slight modification.

Techniques: Activation Assay, Immunohistochemistry, Staining, Expressing

Journal: Journal of translational medicine

Article Title: Fusion cytokine IL-2-GMCSF enhances anticancer immune responses through promoting cell-cell interactions.

doi: 10.1186/s12967-016-0799-7

Figure Lengend Snippet: Fig. 2 Effects of IL2-GMCSF on DC 2.4 cell bioactivities. a FD40 engulfment and b cell proliferation of DC2.4 cells under different dosage of IL2- GMCSF or cytokines; c–d maturation of DC 2.4 cells with different cytokine treatments were indicated by expression of CD80/CD86 (e) and MHC-II/ CD83 (f) detected by flow cytometry. e IL-12 and (F) MDC secretion by DC 2.4 cells treated with IL2-GMCSF or GM-CSF were assayed with ELISA. g Western blotting was used to detect the activation of NF-κB p65 subunit in DC2.4 cells treated as above. In comparison of the effects of IL2-GMCSF and the cytokines alone or in combination, IL-2 was used at 820 IU/mL and GM-CSF at 2.5 × 103 IU/mL, corresponding to the activity of the two parts of the fusion protein at 2.5 × 103 IU/mL. blk: blank; BF: IL2-GMCSF fusion protein; 2CK: the combination of GM-CSF and IL-2. *P < 0.05 com- pared with the control without cytokine treatment. These experiments were repeated at least three times with similar results

Article Snippet: The following primary antibodies were used with 1:2000 dilution: phosphorylated-NF-κB p65 (p-p65; Ser536; 93H1), NF-κB p65 (D14E12) XP® (Cell Signaling Technology, Inc., Beverly, MA), and GAPDH (Zhongshan Goldenbridge Biotechnology Co., Ltd. Beijing, China).

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Comparison, Activity Assay, Control

Journal: Antioxidants

Article Title: Comparison of the Regenerative Metabolic Efficiency of Lipid Extracts from Microalgae Nannochloropsis oceanica and Chlorococcum amblystomatis on Fibroblasts

doi: 10.3390/antiox13030276

Figure Lengend Snippet: Effect of Chlorococcum amblystomatis ( C.a. ) or Nannochloropsis oceanica ( N.o. ) microalgae extracts on the level of subunits of transcription factor NF-κB (p52 and p65) and cytokine TNFα in the following fibroblast groups: o control (n = 5): control cells without and after UVA irradiation [20 J/cm 2 ]; o o C.a. (n = 5): cells cultured for 24 h with C.a. [2 μg/mL] without irradiation and after irradiation with UVA [20 J/cm 2 ]; o N.o. (n = 5): cells cultured for 24 h with N.o. [2 μg/mL] without irradiation and after irradiation with UVA [20 J/cm 2 ]. Mean values ± SD and statistically significant differences for p < 0.05 are presented: a —vs. control; c —UVA+algae N.o. vs. UVA+algae C.a. ; x —vs. UVA; y —UVA+algae C.a. vs. algae C.a. ; z —UVA+algae N.o. vs. algae N.o .

Article Snippet: The levels of the selected proteins NF-κB p65, (OriGene, Rockville, MD, USA); TNFα (Merck, Darmstadt, Germany), NF-κB p52 (LSBio, Seattle, WA, USA), HO-1 (Enzo Life Sciences, Farmingdale, NY, USA), Keap1 (Sino Biological Europe, Düsseldorfer, Eschborn, Germany) and Nrf2 (Gentaur BV, Kampenhout, Belgium) were assessed using the ELISA method [ ].

Techniques: Control, Irradiation, Cell Culture, Algae

Journal: Molecular medicine reports

Article Title: Helicobacter pylori infection enhances heparanase leading to cell proliferation via mitogen‑activated protein kinase signalling in human gastric cancer cells.

doi: 10.3892/mmr.2018.9558

Figure Lengend Snippet: Figure 2. H. pylori infection mediates the HPA protein expression increase in MKN‑45 cells via the MAPK signalling pathway. (A) Western blotting was used to detect the expression levels of p‑p38 MAPK, p38 MAPK, p‑p65 NF‑κB and p65 NF‑κB following different co‑culture durations. (B) p‑p38 MAPK protein expression. (C) p‑p65 NF‑κB protein expression levels were quantitatively analysed using densitometry. (D) Western blots and (E) quantitatively analysed expression of NF‑κB in MKN‑45 cells pre‑treated with SB203580 prior to co‑culture with H. pylori. *P<0.05 and **P<0.01, as indicated. MAPK, mitogen‑activated protein kinase; NF‑κB, nuclear factor‑κB; p‑, phosphorylated; H. pylori or Hp, Helicobacter pylori.

Article Snippet: Following the blocking step, the membranes were incubated with the following primary antibodies: anti-HPA1 (1:1,000; cat. no. ab128931), anti-phosphorylated (p)-p38 MAPK (1:1,000; cat. no. ab195049), anti-p38 MAPK (1:1,000; cat. no. ab170099), anti-p-p65 NF-κB (1:1,000; cat. no. ab76302), anti-p65 NF-κB (1:1,000; no. ab32536; all Abcam, Cambridge, UK) and anti-β-actin (1:2,000; cat. no. TA-09; OriGene Technologies, Inc., Beijing, China).

Techniques: Infection, Expressing, Western Blot

Journal: BMC Complementary Medicine and Therapies

Article Title: Herbal melanin modulates PGE2 and IL-6 gastroprotective markers through COX-2 and TLR4 signaling in the gastric cancer cell line AGS

doi: 10.1186/s12906-023-04124-3

Figure Lengend Snippet: HM-induced IL-6 production and secretion through TLR4 signaling and COX-2 generation by the gastric AGS cells. A Representative Western blot showing the optimization of the concentration of the TLR4 signaling pharmacological inhibitor TAK242 blocking TLR4 signaling, based on the loss of phosphorylation of p65 NFκB, its main downstream target. α-Tubulin was used as a loading control. Bar graph indicates the relative expression of phospho-p65 NFκB, calculated as a ratio of the expression of p65 NFκB. B Optimization of the concentration of the COX-2 pharmacological inhibitor for the blockade of COX-2 production, based on the decrease of COX-2 expression. Bar graph indicates the relative expression of COX-2, calculated as a ratio of the expression of α-Tubulin. C Representative Western blot and bar graph showing and summarizing the impact of the blockade of TLR4 signaling (using TAK242) and of COX-2 inhibitor (NS-398) on LPS- and HM-induced COX-2 and IL-6 production in the AGS cells after 72 h incubation, as compared with LPS- and HM-induced COX-2 and IL-6 production in AGS cells-pretreated with DMSO (solvent used for TAK242 and NS-398 reconstitution). D Bar graph showing the impact of the blockade of TLR4 signaling (using TAK242) and of COX-2 inhibitor (NS-398) on the LPS- and HM-induced secreted PGE2 and IL-6 released by AGS cells after 72 h incubation, detected in the conditional media using an ELISA. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control, from three independent experiments

Article Snippet: The Western blot technology and analysis were done as described in [ ] for the detection of COX-2, IL-6, TLR4, phospho-p65 NFκB, and total p65 NF-κB using goat anti-COX-2 antibody (#AF4198, R&D systems, Minneapolis, MN, USA), rabbit polyclonal anti-IL-6 antibody (#TA328217, OriGene Technologies Inc., Rockville, MD, USA), rabbit monoclonal anti-TLR4 antibody (#ab13867, Abcam, Cambridge, UK), rabbit monoclonal phospho-p65 (Ser 529) NF-κB (#44711G, Invitrogen, Thermo Fischer Scientific) and mouse monoclonal anti-p65 NF-κB (#sc-8008, Santa Cruz Biotechnology), respectively.

Techniques: Western Blot, Concentration Assay, Blocking Assay, Phospho-proteomics, Control, Expressing, Incubation, Solvent, Enzyme-linked Immunosorbent Assay