ngf neutralising antibody  (Alomone Labs)

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Mkp1 is a c-Jun target gene that antagonizes JNK-dependent apoptosis in sympathetic neurons

doi: 10.1523/JNEUROSCI.2824-10.2010

Figure Lengend Snippet: c-Jun and ATF2 bind to two conserved ATF sites in the mkp1 promoter. A, Neuronally differentiated PC12 cells were cultured in the presence (+N) or absence (−N) of NGF for 16 hours. The cells were then cross-linked, chromatin prepared and chromatin immunoprecipitation (ChIP) assays performed with antibodies against Bim (a negative control), c-Jun, ATF2, phospho-c-Jun (serine 63) or phospho-ATF2 (threonine 71). The mkp1 promoter contains two conserved ATF sites (Figure 5) and the ChIP samples (as indicated) and 1% of the input chromatin samples were analysed by PCR using primers that flank the region containing both of the ATF sites. As a negative control, a PCR reaction without chromatin was also tested (H2O). B, Sympathetic neurons were cultured for 7 days in vitro and then refed with medium containing NGF (+N) or anti−NGF antibody (−N). Whole cell extracts were prepared sixteen hours later. EMSA experiments were performed using oligonucleotides containing either the wild type mkp1 ATF site 2 or mutant site, in which four nucleotides have been changed. 4 μg of +N or −N whole cell extract was used per binding reaction as indicated. A control antibody against Bim or c-Jun, ATF2, phospho-c-Jun (ser63) or phospho-ATF2 (Thr71) antibodies were added as shown. The binding reactions were separated on a 5% polyacrylamide gel. Free probe and the AP-1 complexes are indicated. The four point mutations inhibited the binding of AP-1 proteins to the mkp1 ATF site. The c-Jun, ATF2, phospho-c-Jun and phospho-ATF2 antibodies all caused a supershift of the AP-1 complexes and these are indicated (SS).

Article Snippet: For NGF withdrawal experiments, neurons were washed twice in SCG medium lacking NGF and then refed with SCG medium supplemented with a neutralising anti-NGF antibody at 100 ng/ml (Chemicon Europe Ltd, UK).

Techniques: Cell Culture, Chromatin Immunoprecipitation, Negative Control, In Vitro, Mutagenesis, Binding Assay

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Mkp1 is a c-Jun target gene that antagonizes JNK-dependent apoptosis in sympathetic neurons

doi: 10.1523/JNEUROSCI.2824-10.2010

Figure Lengend Snippet: The two conserved ATF sites in the mkp1 promoter contribute to basal promoter activity and are required for promoter induction after NGF withdrawal in sympathetic neurons. A, A 1 kb DNA fragment containing the rat mkp1 promoter was cloned upstream of the Firefly luciferase gene in pGL3-basic. The two conserved ATF sites were then mutated using the QuikChange IIXL site-directed mutagenesis kit. The wild type and mutant mkp1-LUC constructs (10 ng/μl) were microinjected into sympathetic neurons together with the Renilla luciferase construct pRL-TK (5 ng/μl). The injected cells were then refed with medium containing or lacking NGF, as indicated. After 16 hours, a dual luciferase assay was performed and normalised activity was calculated relative to +NGF (grey bars). Statistical significance was calculated relative to +NGF (grey bars) for each construct. The data shown represents the mean of 4 experiments ± SEM. B, Sympathetic neurons were microinjected with wild type or the 2x ATFmutant mkp1-LUC (20 ng/μl), pRL-TK(5 ng/μl) and wild type or kinase dead MLK3 expression vectors (20 ng/μl) or pcDNA3 as a control. Cells were maintained in medium containing NGF for 16 hours and then luciferase activity was measured. Luciferase activity was calculated relative to the level for mkp1-LUC co-injected with pcDNA3 (control), which was set as 1. Statistical significance was calculated relative to pcDNA3 (control) for each construct. The mean of three independent experiments ± SEM is shown. C, Sympathetic neurons were co-microinjected with mkp1-LUC (20 ng/μl) and pRL-TK(5 ng/μl) together with wtMLK3 or kdMLK3 (20 ng/μl) in the presence and absence of mkp1 siRNA (3 μM). Cells were maintained in medium containing NGF for 16 hours and then luciferase activity was measured. Luciferase activity was calculated relative to the level for mkp1-LUC co-injected with the kdMLK3 construct and NTC siRNA. The mean of three independent experiments ± SEM is shown. D, Expression of the JIP-1 JBD reduces induction of an mkp1 reporter construct after NGF withdrawal. Sympathetic neurons were microinjected with mkp1-LUC (20 ng/μl), pRL-TK (5 ng/μl) and pcDNA3 or pcDJBD (50 ng/μl). Cells were maintained + NGF or −NGF for 16 hours and then luciferase activity was measured. Luciferase activity was calculated relative to the level for mkp1-LUC injected with pcDNA3 +NGF, which was set as 1. Statistical significance was calculated relative to +NGF (grey bars) for each construct. The mean of three independent experiments ± SEM is shown. E, Sympathetic neurons were cultured for 6 days in vitro and then microinjected with mkp1-LUC (20 ng/μl), pRL-TK (5 ng/μl) and the c-Jun (H-79) or ATF2 (C-19) antibodies or rabbit immunoglobulin as a control (1 μg/μl) as indicated. Following injection, the cells were maintained in medium containing NGF (+) or anti−NGF antibody (−) for 16 hours before luciferase activity was measured. For each experiment, the level of normalised luciferase activity for control IgG +NGF was set as 1 and other values were calculated relative to this. Statistical significance was calculated relative to +NGF (grey bars) for each antibody co-injection. The mean ± SEM for five independent experiments is shown. Students t-test was used to determine whether inductions were significant: #P <0.001; *P <0.01; +P <0.1, NS (not significant) P > 0.1.

Article Snippet: For NGF withdrawal experiments, neurons were washed twice in SCG medium lacking NGF and then refed with SCG medium supplemented with a neutralising anti-NGF antibody at 100 ng/ml (Chemicon Europe Ltd, UK).

Techniques: Activity Assay, Clone Assay, Luciferase, Mutagenesis, Construct, Injection, Expressing, Cell Culture, In Vitro