Journal:

Article Title: NHERF2 Specifically Interacts with LPA 2 Receptor and Defines the Specificity and Efficiency of Receptor-Mediated Phospholipase C-?3 Activation

doi: 10.1128/MCB.24.11.5069-5079.2004

Figure Lengend Snippet: NHERF2 couples LPA2 to PLC-β3 specifically. (A) LPA2 forms a molecular complex with PLC-β3 in a NHERF2-dependent manner. The NHERF2 constructs (NHERF2, NHERF2 WT; NHERF2 ΔPDZ2, the second PDZ domain-deleted form) were cotransfected in combination with either Flag-PLC-β3 (left) or Flag-PLC-β1 (right) as indicated. After 2 days, COS-7 cells were washed and incubated with PBS containing 0.5 mM DSP (Pierce), which is a cell-permeable cross-linker, for 30 min. After the residual DSP was blocked with PBS containing 50 mM Tris buffer, the cleared lysates were subjected to a pull-down assay using GST-LPA2-CT immobilized onto GSH beads. After a washing step, the resulting precipitates were subjected to SDS-PAGE and then analyzed by Western blot analysis with anti-Flag antibody (top) and anti-NHERF2 antibody (middle) or by Ponceau S staining (bottom) as indicated. The results are representative of three independent experiments. (B) Specific coimmunoprecipitation of PLC-β3 and not of PLC-β1 with Flag LPA2. HeLa cells (at a density of 6 × 106 cells/150-mm dish) were infected with either the recombinant Flag-LPA2 adenovirus or control empty virus at an MOI of 10. At 24 h postinfection, the infected cells were incubated with PBS containing 0.5 mM DSP for 30 min. After the cell lysates were blocked with 50 mM Tris buffer, they were immunoprecipitated with anti-Flag antibody. After a washing step, the resulting precipitates were analyzed with the specific antibodies as indicated. (C) Gene silencing of PLC-β isoforms with specific siRNAs. HeLa cells were transfected with siRNAs directed against PLC-β1 or PLC-β3, along with control siRNA (Luciferase), as described in Materials and Methods. After 3 days, the HeLa cells were lysed and then analyzed by Western blot analysis with specific antibodies, as indicated. (D) PLC-β3 is functionally coupled to LPA2. At 24 h after siRNA transfection, the cells were split at a density of 2 × 105 cells/35-mm well and infected with recombinant adenovirus expressing Flag-LPA2 for 4 h. At 12 h after viral infection, the infected cells were labeled with 1 μCi of [3H]inositol/ml for 12 h and treated with either 0.1% BSA only (−) or 1 μM LPA-BSA conjugates (+). The generation of [3H]IPs was analyzed as described in Materials and Methods. The results are presented as means ± the SE of three experiments performed in duplicate.

Article Snippet: In addition, the specific antibodies to PLC-β1, PLC-β3, and phosphorylated extracellular signal-regulated kinase (ERK) were obtained from the Santa Cruz Co., and the anti-COX-2 antibody was acquired from Caymen Chemicals (Ann Arbor, Mich.).

Techniques: Construct, Incubation, Pull Down Assay, SDS Page, Western Blot, Staining, Infection, Recombinant, Immunoprecipitation, Transfection, Luciferase, Expressing, Labeling

Journal: Food Science & Nutrition

Article Title: Dihydroquercetin Attenuates Silica‐Induced Pulmonary Fibrosis by Modulating the Gut Microbiota and the Serum Metabolites in Mice

doi: 10.1002/fsn3.71389

Figure Lengend Snippet: DHQ treatment attenuated silica‐induced pulmonary fibrosis in C57/BL6 mice. (A, B) DHQ treatment increased the body weight and decreased pulmonary index in silicosis model mice. The changes of body weight (C) The levels of pro‐inflammatory cytokines (IL‐1β, TNF‐α, and TGF‐β) in serum from different groups at day 21 were detected by ELISA assay. (D) Representative pictures (×200) of HE‐stained and Masson‐stained lung sections from mice on day 21 were shown. Bar = 100 μm. (E, F) The inflammation and fibrosis score numbers of 0–3, corresponding to the grades of –, +, ++, and +++, were evaluated by experienced pathologists in a blinded fashion. (G) Representative results of western blot for α‐SMA, collagen I and fibronectin in lung tissues and the quantification of results. Data are shown as mean ± SD. All experiments were repeated three times. # p < 0.05, ## p < 0.01 vs. the control group; * p < 0.05, ** p < 0.01 vs. the SiO 2 group.

Article Snippet: Sigma‐Aldrich provided the SiO 2 (Cat#S5631) particles (around 80% diameter 1‐5 μm), which were filtered through sedimentation following Stokes' law, underwent acidic hydrolysis, and were baked overnight at 200°C for 16 h. Interleukin‐1β (IL‐1β) (CSB‐E08054m), tumor necrosis factor‐α (TNF‐α) (CSB‐E04741m), and transforming growth factor‐β (TGF‐β) (CSB‐E04726m) were acquired from Cusabio Biotechnology in Wuhan, China.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Control