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:

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: Schematic view of the NHERF2-dependent regulation of LPA2-mediated PLC-β3 signaling. Prior to agonist stimulation, both LPA2 and PLC-β3 directly interact with NHERF2, which is localized to the plasma membrane. NHERF2, which is multimerized by a PDZ-PDZ interaction, clusters LPA2 and PLC-β3 in close proximity, thereby creating spatially compact signaling complexes beneath the plasma membrane. Consequently, the LPA2-NHERF2-PLC-β3 complex enables LPA2 to transduce its signal to PLC-β3 with efficiency and specificity.

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: Transduction

Journal: Experimental cell research

Article Title: Regulation of the AGEs-induced inflammatory response in human periodontal ligament cells via the AMPK/NF-κB/ NLRP3 signaling pathway.

doi: 10.1016/j.yexcr.2024.113999

Figure Lengend Snippet: Fig. 3. Indispensability of RAGE in Inflammasome Activation. (A) Transcript expression of RAGE in control HPDLCs versus HPDLCs stimulated with AGEs for 24 h. (B) Protein expression (left) and quantitative analysis (right) of RAGE in control group compared to HPDLCs post 6, 8, and 24 h of culture with AGEs. (C) and (D) A noteworthy diminution in both gene and protein expression of RAGE was observed in RAGE siRNA group as opposed to control group or control siRNA group. (E) Western Blot outcomes and quantitation of NLRP1, NLRP3, and ASC post 48 h of AGEs stimulation with or without RAGE silencing. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In the pursuit of targeted gene silencing, human periodontal ligament cells (HPDLCs) were transfected with RAGE-specific siRNA (Santa Cruz Biotechnology, sc-156124, Dallas, TX, USA) at a concentration of 116.7 nmol/L.

Techniques: Activation Assay, Expressing, Control, Western Blot, Quantitation Assay

Journal: Theranostics

Article Title: Morphine- and foot shock-responsive neuronal ensembles in the VTA possess different connectivity and biased GPCR signaling pathway

doi: 10.7150/thno.90792

Figure Lengend Snippet: The phosphorylated-AKT1 Thr308 level is relatively higher in Shock-Ens, while the expression of PLCβ3 is relatively higher in Mor-Ens. (A) Left, schematic of the experimental procedure for the in-situ proximity ligation assay. Right, the working principal diagram of PLA. (B, D) Representative images of PLA signaling for phosphor-AKT1 Thr308 or PLCβ3 in the VTA. Green: EGFP; Red: p-AKT1 or PLCβ3. Scale bar: left 100 μm, right 25 μm. Dashed lines: outline of EGFP + cell. (C, E) The violin plot and cumulative probability curves illustrate the distribution of the PLA signal intensity in each EGFP + cell. [Mann-Whitney test: (C) Left, Shock-Ens n = 822 cells from 5 mice, Mor-Ens n = 754 cells from 5 mice, U = 240371, P < 0.001; (E) left, Shock-Ens n = 1340 cells from 8 mice, Mor-Ens n = 1415 cells from 8 mice, U = 898116, P = 0.017. Kolmogorov-Smirnov test: (C) right, D = 0.179, P < 0.001; (E) right, D = 0.056, P = 0.026]. *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as mean ± SEM.

Article Snippet: Primary antibodies: Anti-PLCβ3 Antibody (1:200, AP61288, Abcepta Biotech, USA), phosphor-AKT1 Thr308 antibody (1:200, AP67522, Abcepta Biotech, USA).

Techniques: Expressing, In Situ, Proximity Ligation Assay, MANN-WHITNEY