Journal: FEBS Open Bio

Article Title: Complement C5a‐triggered differentiated HL‐60 stimulates migration of THP‐1 monocytic leukocytes via secretion of CCL2

doi: 10.1002/2211-5463.13144

Figure Lengend Snippet: C5a induced phosphorylation of p65 NF‐κB in dHL‐60. (A) Western blot analysis for p‐p65, p‐p38, and p‐ERK in dHL‐60 treated with C5a in a time‐dependent manner. The images represent clopped membranes. The lower graph shows the relative level of phosphorylation relative to the nonphosphorylated form as one. Data presented as the mean ± SD. * P < 0.05 by Dunnett’s test after one‐way ANOVA. (B) mRNA level of CCL2 in C5a‐stimulated dHL‐60 treated with or without the IκB kinase inhibitor, BAY 11‐7082. Data presented as the mean ± SD. * P < 0.05; by two‐tailed unpaired Student’s t ‐test.

Article Snippet: Membranes were reacted with anti‐phospho (p)‐NF‐κB p65, NF‐κB p65, p‐ERK1/2, ERK1/2, p‐p38 MAPK, and p38 MAPK (Santa Cruz Biotechnology) following anti‐mouse or anti‐rabbit secondary antibody conjugated with horseradish peroxidase (GE Healthcare UK Ltd.).

Techniques: Phospho-proteomics, Western Blot, Two Tailed Test

Journal: Cell reports

Article Title: IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages

doi: 10.1016/j.celrep.2018.09.002

Figure Lengend Snippet: (A) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with 100 ng/mL LPS for the indicated times, and cytosolic and nuclear fractions were subjected to western blot analysis for phos-pho-p65/NF-κB (Ser536), phospho p38 and MAPK (Thr180/Tyr182), p105/NF-κB, and p50/NF-κB. GAPDH and heterogenous nuclear ribonucleoprotein L (hnRNPL) were detected as positive controls for cytosolic and nuclear fractions. (B) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with 100 ng/mL LPS for the indicated times, and whole-cell lysates were subjected to western blot analysis for phospho-IRF3 (S386), phospho-STAT1 (Tyr701), IFIT1, and actin (loading control). (C) WT and Ifit1 −/− BMDM cells were stimulated with 100 ng/mL LPS for the indicated times, and whole-cell lysates were subjected to western blot analysis for phospho-STAT1 (Tyr701), phospho-p38 and MAPK (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-ERK (Thr202/Tyr204), and GAPDH (loading control). (D) WT and Ifit1 −/− mouse BMDM transfected with non-targeting control (NTC) or IFIT3 siRNA were stimulated with 100 ng/mL LPS and Ifnb1 mRNA was measured by qPCR. Data are representative of three independent experiments. Data in (D) are expressed as mean ± SD; **p < 0.01, ***p < 0.001 (two-way ANOVA followed by Sidak’s multiple comparison test). See also .

Article Snippet: Following transfer of the proteins, the nitrocellulose membrane was blocked in 5% milk for 1h and probed with the following antibodies overnight at 4°C: goat anti-IFIT1 (Santa Cruz, 82946), Rabbit anti-phos-pho-p38 (Cell Signaling, 4511S), Rabbit anti-phospho-p65 (Cell Signaling, 3033S), Rabbit anti-p105 (Santa Cruz, sc293141), Mouse anti-GAPDH (Cell Signaling, 97166), Rabbit anti-hnRNPL (Santa Cruz, sc-32317), Rabbit anti-phospho-IRF3 (Abcam, ab76493), Rabbit anti-phospho-JNK (Cell Signaling, 9255), Rabbit anti-phospho-ERK (Cell Signaling, 4370), Rabbit anti-phospho-STAT1 (Cell Signaling, 9167S).

Techniques: Control, shRNA, Expressing, Western Blot, Transfection, Comparison

Journal: Cell reports

Article Title: IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages

doi: 10.1016/j.celrep.2018.09.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Following transfer of the proteins, the nitrocellulose membrane was blocked in 5% milk for 1h and probed with the following antibodies overnight at 4°C: goat anti-IFIT1 (Santa Cruz, 82946), Rabbit anti-phos-pho-p38 (Cell Signaling, 4511S), Rabbit anti-phospho-p65 (Cell Signaling, 3033S), Rabbit anti-p105 (Santa Cruz, sc293141), Mouse anti-GAPDH (Cell Signaling, 97166), Rabbit anti-hnRNPL (Santa Cruz, sc-32317), Rabbit anti-phospho-IRF3 (Abcam, ab76493), Rabbit anti-phospho-JNK (Cell Signaling, 9255), Rabbit anti-phospho-ERK (Cell Signaling, 4370), Rabbit anti-phospho-STAT1 (Cell Signaling, 9167S).

Techniques: Virus, Recombinant, Microarray, shRNA, Software

Journal:

Article Title: Analysis of apoptotic and antiapoptotic signalling pathways induced by Helicobacter pylori

doi:

Figure Lengend Snippet: (A) Detection of stable MKN45 transfectants expressing IκBα (SS32/36AA). The clone was analysed using a monoclonal antibody to FLAG and polyclonal antibody to IκBα. (B) Specific protein binding activities of nuclear factor kappa B (NFκB) sequences (electrophoretic mobility shift assay). The nuclear extracts were prepared from MKN45 and MKN45 IκBα SR cells. Cells were treated or not treated with Helicobacter pylori (HP) or 10 ng/ml tumour necrosis factor α (TNF-α) for 90 minutes. Nuclear extracts was incubated with 32P labelled oligonucleotide for 30 minutes. Migration of the DNA-protein complex containing NFκB is indicated. This complex was found to be specific, as judged using supershifting antibody against p65 and cold NFκB probes. (C) MKN45 and MKN45 IκBα SR cells were treated with interferon γ (IFN-γ 10 ng/ml) for 24 hours. Cells were incubated with H pylori (HP) or anti-Fas (CH-11) for 24 hours. Cell viability was assessed by trypan blue dye exclusion assay by counting 300 cells. Viable cells were quantitated under bright field microscopy. Results are expressed as percentages of dead cells. Values are mean (SD) of three independent experiments. *Percentage cytotoxicity was significantly (p<0.05) different between the MKN45 treated cells with anti-Fas or cocultured with H pylori and MKN45 IκBα SR treated cells with anti-Fas or cocultured with H pylori. (D) DNA fragmentation was quantified using a commercially available ELISA (Boehringer Mannheim Biochemicals, Mannheim, Germany): 5×104 cells were incubated in triplicate with H pylori (HP), anti-Fas (CH-11), or medium alone for 12 hours and lysed, and the supernatants were used for ELISA. Absorbance was measured at 405 nm. *Absorbance was significantly (p<0.05) different between the control and treated with anti-Fas or cocultured with H pylori in the MKN45 IκBα SR groups; NS, no significant difference was found. (E) MKN45 and MKN-45 IκBαSR cells were treated with IFN-γ (10 ng/ml) for 24 hours. Cells were incubated with H pylori (HP) or anti-Fas (CH-11) for 12 hours. Immunoblot analysis was performed using anti-caspase-8, caspase-3, cleaved caspase-3, and anti-actin antibody. (F) MKN45 and THP-1 cells were incubated with H pylori and total RNA was extracted at the indicated times. The ribonuclease protection assay was performed according to the supplier’s instructions.

Article Snippet: Detection of NFκB was performed with a 32 P dATP labelled oligo probe containing the NFκB recognition site purchased from Promega (Madison, Wisconsin, USA).

Techniques: Expressing, Protein Binding, Electrophoretic Mobility Shift Assay, Incubation, Migration, Exclusion Assay, Microscopy, Enzyme-linked Immunosorbent Assay, Control, Western Blot