Journal: bioRxiv

Article Title: Sec22b regulates inflammatory responses by controlling the nuclear translocation of NF-κB

doi: 10.1101/2020.09.20.305383

Figure Lengend Snippet: (A) BMDC were incubated with LPS, PGN and zymosan and NO release was assessed by measuring NO 2 - over a period of 2-24 h. Data are presented as mean concentration ± SEM of three independent experiments done in triplicate wells. ***, p < 0.001. (B) The intracellular expression of iNOS (green) in non-stimulated (NS) or LPS-stimulated BMDC was visualized via confocal immunofluorescence microscopy. DNA is in blue; bar, 5 μm. (C) BMDC were stimulated with LPS for 24 h and the colocalization (white pixels, rightmost panels) of iNOS (green) with ER/ERGIC-associated proteins Sec22b, Stx5 (red); ERGIC maker ERGIC53 (red); and Golgi marker P115 (red) was assessed by immunofluorescence. DNA is in blue; bar, 5 μm. Images are representative of three independent experiments. (D) iNOS colocalization was quantified using the Pearson method (see also Figure S1). Data are presented as mean Pearson R coefficient ± SEM of three independent experiments (≥10 cells per experiment); each point represents the coefficient of a single cell.

Article Snippet: 13120, 2982, 9252, 9251, 9212S, 9211, 9102, 9101, 9242, 4764, 6956]; rabbit antibodies anti-Sec22b and -Stx5 from Synaptic Systems [186003, 110053]; rabbit anti-ERGIC53/p58 and mouse anti-β-actin from Sigma [E1031, A1978]; rabbit anti-PDI and -CNX from Enzo Life Sciences [ADISPA890D, ADISPA860D]; rabbit anti-fibrillarin from Abcam [ab4566]; and rabbit anti-P115 from Proteintech [135091AP].

Techniques: Incubation, Concentration Assay, Expressing, Immunofluorescence, Microscopy, Marker

Journal: bioRxiv

Article Title: Sec22b regulates inflammatory responses by controlling the nuclear translocation of NF-κB

doi: 10.1101/2020.09.20.305383

Figure Lengend Snippet: (A) BMDC were fed with opsonised zymosan and recruitment of iNOS (green), Sec22b (red) and Stx5 (red) was visualized via immunofluorescence 24 h post-internalization. Recruitment of LAMP-1 (red) was used as reference. Cytoplasmic and phagosomal (3X-enlarged insets) colocalization can be observed in the rightmost panels (white pixels). DNA is in blue; bar, 5 μm. Images are representative of three independent experiments. (B) Phagosomal recruitment quantification of the aforementioned proteins at 1 h and 24 h post-phagocytosis. Data are presented as mean % positive phagosomes ± SEM of three independent experiments done in triplicate coverslips; each point represents % recruitment in 100 phagosomes per coverslip. *, p < 0.05; ***, p < 0.001; ns, not significant.

Article Snippet: 13120, 2982, 9252, 9251, 9212S, 9211, 9102, 9101, 9242, 4764, 6956]; rabbit antibodies anti-Sec22b and -Stx5 from Synaptic Systems [186003, 110053]; rabbit anti-ERGIC53/p58 and mouse anti-β-actin from Sigma [E1031, A1978]; rabbit anti-PDI and -CNX from Enzo Life Sciences [ADISPA890D, ADISPA860D]; rabbit anti-fibrillarin from Abcam [ab4566]; and rabbit anti-P115 from Proteintech [135091AP].

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Sec22b regulates inflammatory responses by controlling the nuclear translocation of NF-κB

doi: 10.1101/2020.09.20.305383

Figure Lengend Snippet: To evaluate whether Sec22b modulates inflammatory effector secretion, LPS was used to stimulate JAWS-II cells transduced with an shRNA targeting Sec22b (shSec22b) or a scrambled sequence (shScr). Over a period of 24 h, the release of NO (A) TNF (B) and IL-6 (C) was quantified in cell culture supernatants. Data are presented as mean concentration ± SEM of three independent experiments done in triplicate wells. (D) NO secretion in control or LPS-treated RAW264.7 cells transfected with siRNA to a scrambled sequence, Sec22b or Stx5. Data are presented as mean concentration ± SEM of at least two independent experiments done in triplicate wells; each point represents the concentration of a well. (E) iNOS protein levels in non-stimulated (NS) or LpS-stimulated Sec22b-KD JAWS-II cells preincubated with DMSO or brefeldin A (BrefA) were assayed via Western blot. iNOS densitometries were normalized with those of the β-actin loading control, and expressed relative to NS-DMSO-treated cells. Images are representative of two experiments. (F) Inos, Tnf and Il6 mRNA levels in LPS-stimulated Sec22b-KD JAWS-II cells were assayed via RT-qPCR. Data are presented as mean relative expression ± SEM of two independent experiments done in duplicate wells; points represent relative expression in each well. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Article Snippet: 13120, 2982, 9252, 9251, 9212S, 9211, 9102, 9101, 9242, 4764, 6956]; rabbit antibodies anti-Sec22b and -Stx5 from Synaptic Systems [186003, 110053]; rabbit anti-ERGIC53/p58 and mouse anti-β-actin from Sigma [E1031, A1978]; rabbit anti-PDI and -CNX from Enzo Life Sciences [ADISPA890D, ADISPA860D]; rabbit anti-fibrillarin from Abcam [ab4566]; and rabbit anti-P115 from Proteintech [135091AP].

Techniques: Transduction, shRNA, Sequencing, Cell Culture, Concentration Assay, Control, Transfection, Western Blot, Quantitative RT-PCR, Expressing

Journal: The EMBO Journal

Article Title: The innate immune receptor MDA 5 limits rotavirus infection but promotes cell death and pancreatic inflammation

doi: 10.15252/embj.201696273

Figure Lengend Snippet: Micrographs showing the cellular distribution of IRF3 (green) in WT and Ifih1 −/− MEFs detected with an anti‐IRF3 antibody 4 h after infection with RV. Arrows indicate the more intense cytosolic, perinuclear enrichment of IRF3 in the WT and Ifih1 −/− cells before infection. This fluorescence pattern changes to a relatively more intense nuclear (blue) staining of IRF3 in RV‐infected WT cells but not infected Ifih1 −/− cells. The nucleus is visualized with the Hoechst DNA stain.

Article Snippet: Primary antibodies used for Western blots were as follows: mouse anti‐Mda5 (Axxora, ALX‐210‐352), anti‐IκBα (Cell Signaling, 9242), mouse anti‐Il‐1β (Abcam, ab9722), anti‐β‐actin (Abcam, ab8226), mouse anti‐P56 (Dr Ganes C Sen, Cleveland Clinic, USA), and anti‐GFP (SIGMA).

Techniques: Infection, Fluorescence, Staining

Journal: Infection and Drug Resistance

Article Title: Anti-Septic Potential of 7-α-Obacunyl Acetate Isolated from the Toona sinensis on Cecal Ligation/Puncture Mice via Suppression of JAK-STAT/NF-κB Signal Pathway

doi: 10.2147/IDR.S302853

Figure Lengend Snippet: Effects of 7-OBA on JAK2-STAT3 signal pathway in LPS-stimulated RAW264.7 cells. As compared with the control group, ** p <0.01 indicates an extremely significant difference.

Article Snippet: The primary antibodies of p-JAK2 (ab195055), JAK2 (ab39636), p-STAT3 (ab30647), and STAT3 (ab19352) were purchased from Abcam Biotech (Cambridge, MA); NF-κB p65 (#8242), Histone H1 (#41328), and IκB (#9242) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: