Journal: Journal of Cell Communication and Signaling

Article Title: Scutellarin alleviates microglia‐mediated neuroinflammation and apoptosis after ischemic stroke through the PI3K/AKT/GSK3 β signaling pathway

doi: 10.1002/ccs3.12023

Figure Lengend Snippet: Schematic diagram illustrating the regulatory mechanism of scutellarin in microglia‐mediated neuroinflammation and apoptosis in IS. Scutellarin inhibited the NF‐ κ B pathway, pro‐apoptotic proteins Bax and caspase‐3, and inflammatory factors TNF‐ α and IL‐1 β in microglia after IS. It also promoted the activation of the PI3K/AKT/GSK3β pathway and anti‐apoptotic protein Bcl‐2. Conversely, the PI3K pathway inhibitor promoted the expression of the NF‐ κ B pathway, pro‐apoptotic proteins, and inflammatory factors while inhibiting the expression of the PI3K/AKT/GSK3 β pathway and anti‐apoptotic protein Bcl‐2. These results suggest that scutellarin regulates activated microglia‐mediated neuroinflammation and apoptosis via the PI3K/AKT/GSK3 β and NF‐ κ B pathways.

Article Snippet: Protein bands were blotted onto polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk powder for 2 h. After washing with tris buffered saline with tween‐20 (TBST), the membranes were treated respectively with PI3K (Mouse Anti‐IgG, 1:1000; Protein‐tech, USA, #60225‐1‐Ig), p‐PI3K (Rabbit Anti‐IgG, 1:1000; Bioss, Beijing, #bs‐5570R), AKT (Rabbit Anti‐IgG, 1:2000; Protein‐tech, USA, #10176‐2‐AP), p‐AKT (Mouse Anti‐IgG, 1:1000; Protein‐tech, USA, #66444‐1‐Ig), GSK3β (Rabbit AntiIgG, 1:1000; Protein‐tech, USA, #22104‐1‐AP), p‐GSK3β (Rabbit Anti‐IgG, 1:1000; Bioss, Beijing, #bs‐2066R), NF‐κB (Rabbit Anti‐IgG, 1:1000; Protein‐tech, USA, #80979‐1‐RR), p‐NF‐ κ B (Rabbit Anti‐IgG, 1:1000; Bioss, Beijing, #bs‐0982R), TNF‐α (Mouse Anti‐IgG, 1:1000; Protein‐tech, USA, #60291‐1‐Ig), IL‐1β (Rabbit Anti‐IgG, 1:1000; Affinity, Jiangsu, #AF5103), Bax (Mouse Anti‐IgG, 1:1000; Bioss, Beijing, #bs‐0127M), Bcl‐2 (Rabbit Anti‐IgG, 1:1000; Bioss, Beijing, #bs‐0032R), caspase‐3 (Rabbit Anti‐IgG, 1:1000; Protein‐tech, USA, #19677‐1‐AP), and cleaved‐caspase‐3 (Rabbit Anti‐IgG, 1:1000; Affinity, Jiangsu, #AF7022) overnight at 4°C.

Techniques: Activation Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: IRF3-dependent Type I Interferon Response in B Cells Regulates CpG-mediated Antibody Production

doi: 10.1074/jbc.m704755200

Figure Lengend Snippet: FIGURE 6. CpG-mediated Type I IFN response in B cells regulates survival and antibody production. A, Ifnar/- and Ifnar/-purified B cells were stimulated with CpG-B. Surface ICAM-1 expression was analyzed by flow cytometry after 24 h of CpG-B stimulation. Proliferation was assessed by [3H]thymidine incorporation. B, Ifnar/- and Ifnar/-purified B cells were stimulated either with CpG-B or left in media alone. Cells were stained daily with propidium iodide (PI) and analyzed by flow cytometry. The PI-negative population is expressed as percent viable cells. C, After 5 days of stimulation, Bcellsfromindicatedgenotypeswerestainedwithanti-Syndecan-1antibody and analyzed for Syndecan-1 expression by flow cytometry. Isotype control for each sample is shown as a filled curve. D, supernatants from 5-day CpG-B- stimulated B cell cultures were assessed for the presence of IgM by ELISA. Supernatants from 6-day cultures were assessed for the presence of IgG2a by ELISA.Supernatantsfrom9-daycultureswereassessedforthepresenceofIgE by ELISA. Data represent at least two independent experiments. Error bars indicate SD among duplicates.

Article Snippet: 96-well plates were coated with 2 g/ml rabbit anti-mouse IgM, antiIgG (H L), or anti-IgE (Southern Biotechnology) in sodium carbonate buffer, pH 9.6 overnight and blocked with 1% bovine serum albumin (Sigma) for 1 h. After a 1-h incubation with culture supernatants and standards (eBiosciences), plates were washed and further incubated with horse radish peroxidaseconjugated goat anti-mouse IgM, anti-mouse IgG2a, or antimouse IgE (Southern Biotechnology).

Techniques: Purification, Expressing, Flow Cytometry, Staining, Control, Enzyme-linked Immunosorbent Assay