recombinant human pro-ngf  (GenScript corporation)

Journal: The Journal of Clinical Investigation

Article Title: Role of proNGF/p75 signaling in bladder dysfunction after spinal cord injury

doi: 10.1172/JCI97837

Figure Lengend Snippet: (A) ProNGF is released into the urine almost immediately after spinal cord transection in mice. The blots were probed with anti-proNGF and pan-NGF antibodies. There was little mature NGF detectable in the urine at these time points. (B) Quantification of proNGF by Western blotting. Within-subject comparison was significant at P = 0.003 based on repeated measures of 1-way ANOVA after Greenhouse-Geisser correction, which resulted in P < 0.01. Subsequent pairwise comparisons were made using Bonferroni correction. (C) Quantification of proNGF detected in mouse urine by proNGF-specific ELISA. Note that mature NGF was not detected by mature NGF-specific ELISA. (D) Immunoprecipitation/Western analyses of urine samples illustrate that the bands detected with proNGF antibody in A are indeed proNGF. Mouse urine samples were immunoprecipitated with 27/21 mature NGF antibody and probed with proNGF antibody. (E) ProNGF is detected in the urine after contusion injuries in mice. (F) ProNGF is present in the urine after spinal cord transection in rats. Mature NGF was not detected by mature NGF-specific ELISA. (G) ProNGF was also detected in human urine after SCI. The upper blot was probed with proNGF-specific antibody (*artifact), while the lower blot was probed with pan-NGF antibody (H-20). Note that recombinant proNGF and mature NGF were included as controls. Based on human proNGF-specific ELISA, the amount of proNGF was 1 and 46 pg/mg for 3 and 6 hours after injury, respectively. Note that 3- and 6-hour urine samples were collected from 2 different individuals. ELISA assays also failed to detect mature NGF as in Western blotting. C, urine from healthy control without SCI.

Article Snippet: For NGF addition, cells were incubated with 10 ng/ml of recombinant pro-NGF (Alomone) for 30 minutes after Fluo-4 AM loading, imaged for 3 minutes, then treated with 50 nM GSK1016790A followed by 30 minutes of imaging.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Recombinant

Journal: Journal of Neurotrauma

Article Title: Fluoxetine Prevents Oligodendrocyte Cell Death by Inhibiting Microglia Activation after Spinal Cord Injury

doi: 10.1089/neu.2014.3527

Figure Lengend Snippet: Fluoxetine inhibits p38-MAPK activation and pro-NGF production in microglia after spinal cord injury (SCI). Spinal cord from mice treated with fluoxetine at 3 or 5 days after injury was prepared as described in the Methods section (n=3). (A) Presence of p-p38-MAPK–positive microglia in the ventral horn of gray matter at 5 days after injury. Representative images are from sections 1 mm rostral to the lesion epicenter. Scale bar, 50 μm. (B) Western blot of p-p38-MAPK at 3 and 5 days after injury. (C) Densitometric analysis of Western blot. Note that fluoxetine inhibits p38-MAPK activation after SCI, as compared to vehicle control. (D) Real-time reverse-transcriptase polymerase chain reaction of NGF at 3 days after injury. (E) Western blot of pro-NGF at 5 days after SCI. (F) Densitometric analysis of Western blot. Note that fluoxetine inhibits pro-NGF expression at 5 days after SCI. Values are presented as means±standard deviation. *p<0.05 versus vehicle control. d, days; p-p38MAPK, phosphorylated p38 mitogen-activated protein kinase; pro-NGF, pro-nerve growth factor.

Article Snippet: Membranes were blocked with 5% nonfat skim milk or 5% bovine serum albumin in Tris-buffered saline solution with 0.1% Tween 20 for 1 h at room temperature and then incubated with primary Abs against p-p38-MAPK (1:1000; Cell Signaling Technology), p38-MAPK (1:1000; Cell Signaling Technology), pro-NGF (1:1000; Alomone Labs, Jerusalem, Israel), p-c-Jun (1:1000; Cell signaling Technology), c-Jun (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), cleaved caspase-3 (1:1000; Cell Signaling Technology), MBP (1:500; Millipore), and RhoA (1:1000; Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Activation Assay, Western Blot, Polymerase Chain Reaction, Expressing, Standard Deviation