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Inhibition of neurite outgrowth using comme(2)
To detect cytoplasmic internalization of MAG-Fc, cells were harvested at 5, 10, 20, 30, 40, and 50 minutes after addition of MAG-Fc to cells. Immunofluorescence of MAG was quantified by integrated optical density (IOD) using NIH ImageJ software (
For measurement of neurite outgrowth of neuro-2a cells after MAG treatment, a neurite was defined as a process extending from the cell body that was at least longer than the length of the cell. After 24, 48, and 72 hours of MAG-Fc chimera treatment, neuro-2a cells were fixed with 4% paraformaldehyde and then incubated with a rabbit anti-βIII-tubulin primary antibody (1:1000; Boster Biotechnologies, Inc.,Wuhan, China) overnight, at 4°C. Cells were then incubated with a secondary antibody (donkey anti-rabbit IgG conjugated with Alexa Fluorescence-594, 1:500; Life Technologies, Carlsbad, CA, USA) for 1 hour at room temperature to visualize neurite morphology. Neurite outgrowth was measured from immunofluorescence images using ImageJ software. The measurements included the total length of the neurites, the percentage of cells with neurites relative to the total number of cells in each image. Fifteen to twenty images were included for each MAG concentration group. In addition, the best MAG-Fc concentration was chosen upon neurite measurement.
The quantification of neurite outgrowth, the percentage of cells with neurites, and the strength of target protein immuno fluorescence were analyzed using ImageJ software (
Detection of RhoA activity
Detection of RhoA activity was measured by enzyme-linked immunosorbent assay (ELISA). Brie fly, neuro-2a cells were plated into 24-well plates, and 20 nM MAG-Fc was added to the medium, as described above. After 5, 10, 20, or 40 minutes of MAG treatment, the whole cell protein was extracted with radioimmunoprecipitation assay (RIPA) buffer, which contains 1% protease inhibitor (freshly added) and 1% phosphatase inhibitor. Protein concentration was measured by the Braford method (Bradford, 1976). The activity of RhoA was measured using a commercial RhoA activity assay ELISA kit (Jingma Bioscientifics, Inc., Shanghai, China). The procedure was followed according to the manufacturer’s instructions. The activity of RhoA is expressed as U/g.
Detection of ROCK activation induced MAG-Fc by western blot assay
The function of ROCK after MAG-Fc treatment was evaluated by its phosphorylation and translocation in the cytoplasm of neuro-2a cells. The phosphorylation of ROCK was detected by the appearance of phospho-ROCK bands via western blot assays. The level of ROCK phosphorylation was calculated from the IOD value. Translocation of RhoA/Rho kinase and pathway activation, indicating ROCK activation(Zhang and Jin, 2017), was detected by strong immuno fluorescence of ROCK translocating from areas deep in the cytoplasm close to the nucleus toward areas adjacent to the cell membrane.
For sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot assays, whole cell protein of neuro-2a cells incubated with 20 nM MAG-Fc (for 5, 10, 20,and 40 minutes) was extracted with RIPA buffer. Thirty micrograms of protein from each group was loaded onto 10%gels and separated by electrophoresis. After transferring the protein from the gel to a polyvinylidene fluoride membrane(Immobilon-P, EMD Millipore Corporation, Billenca, MA USA), the membrane was incubated with a rabbit anti-phospho-ROCK antibody (1:500; Abcam) overnight at 4°C, then with a goat-anti-rabbit IgG secondary antibody labeled with horseradish peroxidase. The membranes were then stained with EasySee Western Blot Kit Luminous fluid (TransGen Biotech, Beijing, China). Positive phospho-ROCK bands were quantified with ImageJ software. The immuno fluorescence of phospho-ROCK was assessed as described above.
Statistical analysis
All data are presented as the mean ± SE. Statistical signi ficance was determined using GraphPad Prism 5.0 (Graph-Pad software, La Jolla, CA, USA), using one-way analysis of variance followed by t-test. P < 0.05 was regarded as statistically significant.
Results
Visualization and intracellular distribution of exogenous MAG in cultured neuro-2a cells
Cultured neuro-2a cells express extremely low levels of MAG (Figure 1). Intracellular MAG immunofluorescence was observed when 20 nM MAG-Fc was added to the cell medium while plating the cells. The cytoplasmic distribution of MAG was clearly identified after incubation with MAGFc for minutes to hours (10 minutes to 6 hours). Remarkable MAG staining was observed after 10 minutes of exogenous MAG-Fc treatment. The cytoplasmic MAG immunofluorescence tended to increase with MAG-Fc incubation IOD of MAG immunoreactivity was reached 50 and 60 minutes of MAG-Fc treatment (P < 0.05). The IOD value of MAG at 50 and 60 minutes was twice that of controls (P< 0.001). However, longer MAG incubation times did not increase intracellular MAG immunofluorescence further,and MAG staining even decreased for incubation times approaching 6 hours (Figure 1).
文章来源:《新商务周刊》 网址: http://www.xswzkzzs.cn/qikandaodu/2020/1021/526.html