Journal:

Article Title: Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide

doi: 10.1042/BJ20040217

Figure Lengend Snippet: A region flanking the instability site in human plasminogen activator inhibitor type 2 mRNA [25] is also present in human IL-1 family members as IL-1F7b, IL-1β IL-18 and IL-1Ra. Alignment was generated by using LALIGN-program (www.ch.embnet.org) to compare two DNA sequences for local similarity.

Article Snippet: The monoclonal antibodies against human IL-18 (MAB318) and against His 6 -tagged proteins (MAB050) were purchased from R&D Systems (Minneapolis, MN, U.S.A.).

Techniques: Generated

Journal:

Article Title: Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide

doi: 10.1042/BJ20040217

Figure Lengend Snippet: (A) Freshly isolated human monocytes were stimulated or not with LPS (10 μg/ml) for 4 h. After co-staining against IL-1F7b and Il-18, the cells were visualized using confocal digital microscopy. Red dye, anti-IL-1F7b; green dye, anti-IL-18; blue dye, nuclear stain. (B) Fluorescence was recorded for single cells and the mean counts of intensity (±S.E.M.) for IL-1F7b or IL-18 were calculated by analysing at least 70 individual cells. Statistical differences were calculated by ANOVA; ***P<0.0001.

Article Snippet: The monoclonal antibodies against human IL-18 (MAB318) and against His 6 -tagged proteins (MAB050) were purchased from R&D Systems (Minneapolis, MN, U.S.A.).

Techniques: Isolation, Staining, Microscopy, Fluorescence

Journal:

Article Title: Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide

doi: 10.1042/BJ20040217

Figure Lengend Snippet: RAW264.7 cells stably transfected with human IL-18 cDNA were stimulated with LPS (10 ng/ml). IL-18 mRNA and intracellular protein levels were analysed at the indicated times. (A) Semi-quantitative PCR of one representative experiment. (B) Densitometric analysis of six independent experiments (means±S.E.M.). (C) The lysate of transfected RAW264.7/IL-18 cells before and after treatment with LPS (10 ng/ml) was separated by SDS/PAGE (10% gel) and blotted on to nitrocellulose. The blot was stained using a mAb against human IL-18.

Article Snippet: The monoclonal antibodies against human IL-18 (MAB318) and against His 6 -tagged proteins (MAB050) were purchased from R&D Systems (Minneapolis, MN, U.S.A.).

Techniques: Stable Transfection, Transfection, Real-time Polymerase Chain Reaction, SDS Page, Staining

Journal: Molecular cancer therapeutics

Article Title: Systemic checkpoint blockade by PD-L1 single-chain antibody confers potent anti-tumor immunity and long-term survival

doi: 10.1158/1535-7163.MCT-22-0010

Figure Lengend Snippet: (A) Single-chain variable fragments (scFv) against murine programmed death-ligand 1 (PD-L1) was subcloned into a rAAV vector. The expression of PD-L1 scFv was confirmed by transducing rAAV-PD-L1-scFv in HEK 293T cells and detecting high-level expression and extracellular secretion in conditioned media (CM) using an anti-His antibody by Western blot. (B) Splenocytes from OT-1 transgenic mice were plated at a density of 5×105 cells/well in 24-well tissue culture plates. Following overnight culture, cells in replicates were stimulated with the OT-1 peptide (0.5 μg/mL). The bioactivity of PD-L1 scFv from rAAV-PD-L1-scFv-transduced 293T conditioned medium was examined by treating OT-1 peptide-pulsed T cells in the presence or absence of purified recombinant PD-L1 protein (100 ng/ml). Live splenocytes were counted at 24 h intervals to monitor kinetics of splenocytes in response to the peptide stimulation (*p<0.05, **p<0.01, ***p<0.001). (C) Cohorts of C57BL/6 mice were administered with either a one-time intramuscular application of rAAV-PD-L1-scFv (left panel) or a four-time application (once every three days) with 200 μg/mouse biotinylated mouse PD-L1 monoclonal antibody (right panel). Sera were collected from mice on indicated days after completion of a one-time application of rAAV-PD-L1-scFv or PD-L1 mAb applications. The systemic level of PD-L1 scFv was detected by Western blotting using an anti-His-HRP antibody (left panel) and the level of PD-L1 monoclonal antibody was detected using streptavidin-HRP (right panel). Quantitation of PD-L1 scFv and PD-L1 mAb was performed by ELISA.

Article Snippet: Anti-His-HRP monoclonal antibody was purchased from R&D Systems (MAB050H).

Techniques: Plasmid Preparation, Expressing, Western Blot, Transgenic Assay, Purification, Recombinant, Quantitation Assay, Enzyme-linked Immunosorbent Assay