Journal: STAR Protocols

Article Title: Protocol for screening cellular outputs activated by optogenetically controlled temporal PI3K signaling activation patterns

doi: 10.1016/j.xpro.2023.102622

Figure Lengend Snippet:

Article Snippet: Western BLoT Rapid Detect v2.0 , Takara , T7122A.

Techniques: Recombinant, Sterility, Protease Inhibitor, Titration, Plasmid Preparation, Software, Western Blot, Fluorescence, Microscopy

Journal: RMD Open

Article Title: IL-6-PAD4 axis in the earliest phase of arthritis in knock-in gp130F759 mice, a model for rheumatoid arthritis

doi: 10.1136/rmdopen-2018-000853

Figure Lengend Snippet: PAD4 + cells in the synovium of gp130F759 are neutrophils. (A) Amounts of PAD4 protein and STAT3 phosphorylation in the wrist joints from wild type (WT) and gp130F759. Lysates of the joints from three mice of each genotype were pooled and subjected to western blotting. (B) Frozen sections of the synovium from WT and gp130F759 were stained with anti-PAD4 and pSTAT3 antibodies and Cy3-labelled F(ab’) 2 donkey anti-rabbit IgG (H+L). Representative pictures from four independent experiments are shown. Nuclei were stained with Hoechst 33342. Colocalisation was examined with anti-PAD4 and biotinylated pSTAT3 antibodies, which were visualised with Cy3-labelled anti-Rabbit IgG and Alexa488-streptavidin, respectively. A PAD4 producing cell whose STAT3 is phosphorylated (inset). The bars indicate 20 µm. (C) Gene expression levels of Padi4 and Il6 in haematopoietic (CD45 + ) or non-haematopoietic (CD45 − ) synovial cells which were separated with rat anti-mouse CD45 antibody and sheep anti-rat IgG magnetic beads. Summarised data from three independent experiments are shown. (D) IL-6 concentration in supernatant of primary culture of CD45 − synovial cells from WT and gp130F759 at 5 months old (n=7). *p<0.05. (E) Morphology of synovial cells producing PAD4. The photos of representative cytospin specimens from WT and gp130F759 are shown. Synovial cells cytospun onto the slide glass were incubated with anti-PAD4 antibody (red) and Hoechst 33342 for nuclear staining (blue). In the graph, black bar shows average and the open circle shows individual value for each mouse. (F) IL-6 produced by CD45 − synovial cells induced Padi4 expression in neutrophils. WT bone marrow neutrophils were stimulated for 6 hours with culture supernatant fluid (CSF) from gp130F759 pretreated with anti-IL-6 antibody or control IgG1. Then RNA from the neutrophils were prepared and transcription of Padi4 was estimated by real-time PCR using specific primers and SYBR green. Relative expression levels compared with Actb are shown. (G) IL-6 in the CSF from gp130F759 induced activation of STAT3 in neutrophils. WT bone marrow neutrophils were stimulated for 30 min with CSF from gp130F759 pretreated with anti-IL-6 antibody or control IgG1. The neutrophils were cyto-spun, air-dried and stained with anti-pSTAT3 antibody and Cy3-labelled donkey anti-rabbit IgG antibody. Pictures were taken with LSM700 confocal microscope. The bars indicate 10 µm.

Article Snippet: Anti-pSTAT3 antibody was stripped with Western BLoT Stripping Buffer (TAKARA Bio, Shiga, Japan), and then the membrane was reprobed with rabbit anti-STAT3 antibody (Cell Signaling Technology).

Techniques: Western Blot, Staining, Expressing, Magnetic Beads, Concentration Assay, Incubation, Produced, Real-time Polymerase Chain Reaction, SYBR Green Assay, Activation Assay, Microscopy