Anti ngf antibody

Undifferentiated PC12 cells were plated onto collagen IV-coated plates (Corning) and differentiated in culture medium containing 50 ng/ml NGF (Accurate Chemical) for 5 – 7 days. Primary sympathetic neuroblasts isolated from SCGs of post-gestation rat pups (P2) were cultured as previously described [20 (link)]. Differentiated PC12 cells and neuroblasts were infected with lentivirus encoding shXAF1 or nontargeting control virus (shSCR) followed by NGF withdrawal with anti-NGF antibody (1:5000, Sigma) for up to 48 hours where they were harvested for subsequent analysis.

Undifferentiated PC12 cells were plated onto collagen IV-coated plates (Corning) and differentiated in culture medium containing 50 ng/ml NGF (Accurate chemicals) for 5–7 days as described^{20 (link),21 (link)}. Differentiated PC12 were pre-treated with 10 mM N-acetylcysteine (NAC) (Sigma Aldrich) before NGF withdrawal and continuously replenished every 12 hours after NGF withdrawal with anti-NGF antibody (1:5000, Sigma Aldrich) for up to 48 hours where they were harvested for Immunoblot analysis.

On postnatal days 3–14, pups were removed from their home cage daily for 3 h consecutively (9:00 am to 12:00 pm). After separation, NMS pups were returned to their mothers’ cages and left undisturbed, whereas control pups were nursed as usual. All pups were weaned on postnatal day 22. To investigate the involvement of NGF/TrkA signaling in NMS response, pups were daily administered with/without recombinant NGF (1 μg/kg, Thermo Fisher), anti-NGF antibody (1 mg/kg, Sigma), K252a (50 μg/kg, Sigma), NMAC13 (100 μg/kg, Absolute Antibody), or IWP-2 (20 μM, Sigma) by intraperitoneal injection. Injection of control IgG or dimethyl sulfoxide served as a control. For most of the experiments, animals were killed for phenotypic analyses at 8 weeks upon maternal deprivation. To examine the impact of IWP-2-mediated Wnt inhibition on NGF/NMS-induced intestinal changes, the mice were killed for analyses at the 16 days of age. The diagram showing the timeframe for the experiment is shown in (Supplementary Fig. 14). The animal care and sample analysis were not blinded to the group allocation in the animal experiments.