Journal:

Article Title: Anabolic effects of a G protein-coupled receptor kinase inhibitor expressed in osteoblasts

doi: 10.1172/JCI14663

Figure Lengend Snippet: Expression of the GRK2-CT enhances PTH/PTHrP receptor responsiveness and inhibits agonist-induced phosphorylation of the PTH/PTHrP receptor. HEK293 cells were cotransfected with the rat PTH/PTHrP receptor cDNA and either the GRK2-CT or empty vector. Two days after transfection, expression of GRK2 and the GRK2-CT was assessed by immunoblotting as described in Methods using an Ab that recognizes the C terminus of both GRK2 and GRK3 (25). In parallel experiments, we investigated the effect of the GRK2-CT on PTH/PTHrP receptor responsiveness by measuring cAMP generation and agonist-induced phosphorylation of the PTH/PTHrP receptor by immunoprecipitation of 12CA5-tagged PTH/PTHrP receptors, as described in Methods. Apparent molecular mass is indicated in kilodaltons. (a) In lane 1, HEK293 cells express predominantly GRK2. A slightly smaller protein corresponding to the GRK3 was detected by immunoblotting only after prolonged exposure of the radiographic film. Lane 2 is a positive control prepared from HEK293 cells transfected with the GRK2 cDNA. (b) Cotransfection of the GRK2-CT significantly enhanced PTH/PTHrP receptor responsiveness compared with cells cotransfected with empty vector. The inset shows that the increase in PTH/PTHrP receptor responsiveness was associated with high levels of GRK2-CT expression in this model system. (c) Cotransfection of the GRK2-CT inhibited agonist-induced phosphorylation of the PTH/PTHrP receptor. (d) The amount of PTH/PTHrP receptor in the immunoprecipitates was assessed by immunoblotting using the 12CA5 Ab. Similar amounts of PTH/PTHrP receptor were immunoprecipitated from cells cotransfected with either the GRK2-CT or empty vector. *P < 0.05 vs. vector.

Article Snippet: For each transfection, 1 ml of transfection solution was added (in drops) to a single 60-mm dish or to two wells of a six-well plastic culture dish (9.5 cm 2 /well) (Corning-Costar Corp.).

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Immunoprecipitation, Positive Control, Cotransfection

Journal: Cell Death & Disease

Article Title: Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

doi: 10.1038/cddis.2017.58

Figure Lengend Snippet: Phosphorylation of RIP1 switched necroptosis in cultured neurons in model of ICH in vitro . As phosphorylation of RIP1 has an essential role of necroptosis, we used overexpression and with a mutation of phosphorylation site (S166A) of RIP1 to investigate its role in necroptosis in ICH. ( a ) Flow cytometry indicated that, compared with the conditioned medium group, it turned up a higher ratio of necroptosis (~66.7%) and a lower ratio of apoptosis (~0.9%) in neurons during overexpression of RIP1 by Ad-RIP1 transfection. Besides, this phenomenon could be significantly reduced in the S166A group (necroptosis ~34.2%) and in the Nec-1 treatment group (necroptosis ~21.7%). ( b ) Related bar graph showed four different conditions of neurons that had been stated above. ** P =0.0001 in PI+/Annexin V− cells versus the conditioned medium group; && P =0.0027 in PI+/Annexin V− cells versus the Ad-RIP1 group; $$ P <0.0001 in PI+/Annexin V− cells and $$ P <0.0001 in PI−/Annexin V+ cells versus the Ad-RIP1 group; all were unpaired t-test, n =3. ( c ) IP demonstrated that interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were increased in the Ad-RIP1 group. These increased interactions could be attenuated remarkably in the S166A group. ( d ) Consistent data analysis of IP. ** P =0.0015 in p-Ser, ** P =0.0004 in RIP3, ** P =0.0013 in MLKL and ** P =0.0003 in caspase-8 versus the conditioned medium group; & P =0.0493 in p-Ser, & P =0.0126 in RIP3, && P =0.0036 in MLKL and && P =0.0018 in caspase-8 versus the Ad-RIP1 group. The mean values for the control group were normalized to 1.0; all were unpaired t -test, n =3. ( e ) PI and Hoechst staining indicated that the ratio of necroptosis upregulated (as arrows point to, PI+/Hoechst+ cells) in the overexpression group, whereas mutation of the phosphorylation site inhibited this effect. Scale bar=50 μ m. ( f ) Numbers of PI+/Hoechst+ cells, ** P <0.0001 versus the conditioned medium group; && P <0.0001 versus Ad-RIP1 group; all were unpaired t -test, n =6. All data are expressed as means±S.E.M.

Article Snippet: All of them were stored at −80 °C and diluted to 1 × 10 9 PFU/ml in an enhanced transfection solution (Genescript) before intracerebroventricular injection in vivo and diluted to 1 × 10 8 PFU/ml before being transfected to the cultured neuron.

Techniques: Phospho-proteomics, Cell Culture, In Vitro, Over Expression, Mutagenesis, Flow Cytometry, Transfection, Control, Staining