human neuroglobin (ngb)  (GenScript corporation)

Journal: Cell Death & Disease

Article Title: Neuroglobin promotes neurogenesis through Wnt signaling pathway

doi: 10.1038/s41419-018-1007-x

Figure Lengend Snippet: NPC was cultured in differentiation medium (DMEM/F12 + 2%FBS) and Lentivirus containing Ngb or GFP gene was transduced on day 2 of culture. Differentiated cells were examined by immunocytochemistry using anti-Tuj1 (immature neuron maker) and anti-GFAP antibodies. NPC differentiation was examined by immunostaining for Tuj1 (immature neuron) and GFAP (astrocytes). a Representative images of Tuj1 and GFAP immunostaining; b Quantification of percentages Tuj1 positive cells, c Quantification of average neurite length ( n = 4,* p < 0.05 vs Lv-GFP)

Article Snippet: The blots were reacted with the following primary antibodies: chicken anti-Ngb (1:2000; BioVendor, Candler, NC), mouse anti-Dvl1 (1:1000, Santa Cruz), rabbit anti-β-catenin (1:1000, Abcam), mouse anti-Histone-H3 (1:1000, cell signaling), mouse anti-β-actin (1:5000, Millipore, Billerica, MA) as a control in a solution containing 0.05% Tween-20, 1% bovine serum albumin, and 4% nonfat dry milk at 4 °C overnight.

Techniques: Cell Culture, Immunocytochemistry, Immunostaining

Journal: Cell Death & Disease

Article Title: Neuroglobin promotes neurogenesis through Wnt signaling pathway

doi: 10.1038/s41419-018-1007-x

Figure Lengend Snippet: To investigate the mechanisms of the neurogenesis promotional effect by Ngb overexpression, we examined the protein levels of Dvl1 and β-catenin, the two key components of Wnt signaling, after Lv-Ngb transduction in cultured neurosphere. a Representative Western blot images for Dvl1 and β-catenin using cell lysate of cultured NPCs. β-actin was used as loading control. b Quantification of Dvl1 protein level; c Immunostaining for β-catenin in cultured NPC; d Representative Western blot images and e quantification of β-catenin in isolated nuclear proteins. Histone-H3 was used as nucleus loading control ( n = 4,* p < 0.05 vs Lv-GFP). We further used a Wnt inhibitor, IWR-endo, to test the involvement of Wnt signaling in the neurogenesis promotional effect by Ngb overexpression; f Relative neurosphere number after Lv-Ngb transduction and IWR-endo treatment; g Relative neurosphere size after Lv-Ngb transduction and IWR-endo treatment; h The percentage of Tuj positive cell numbers in NPC differentiation after Lv-Ngb transduction and IWR-endo treatment ( n = 4, * p < 0.05 vs Lv-GFP, # p < 0.05 vs Lv-Ngb)

Article Snippet: The blots were reacted with the following primary antibodies: chicken anti-Ngb (1:2000; BioVendor, Candler, NC), mouse anti-Dvl1 (1:1000, Santa Cruz), rabbit anti-β-catenin (1:1000, Abcam), mouse anti-Histone-H3 (1:1000, cell signaling), mouse anti-β-actin (1:5000, Millipore, Billerica, MA) as a control in a solution containing 0.05% Tween-20, 1% bovine serum albumin, and 4% nonfat dry milk at 4 °C overnight.

Techniques: Over Expression, Transduction, Cell Culture, Western Blot, Immunostaining, Isolation

Journal: Cell Death & Disease

Article Title: Neuroglobin promotes neurogenesis through Wnt signaling pathway

doi: 10.1038/s41419-018-1007-x

Figure Lengend Snippet: To further investigate the involvement of Wnt signaling in Ngb-overexpression-induced neurogenesis, we tested the protein levels of Dvl1 and β-catenin in peri-infarct mice brain at 5 days after stroke. a Representative images of Western blot for Dvl1 and β-catenin from peri-infarct brain lysate; b , c Quantification of Dvl1 and β-catenin; d Representative images of Western blot for β-catenin from isolated nuclear protein; e Quantification of β-catenin in nuclear extract. ( n = 4, * p < 0.05 vs sham, # p < 0.05 vs MCAO Lv-GFP)

Article Snippet: The blots were reacted with the following primary antibodies: chicken anti-Ngb (1:2000; BioVendor, Candler, NC), mouse anti-Dvl1 (1:1000, Santa Cruz), rabbit anti-β-catenin (1:1000, Abcam), mouse anti-Histone-H3 (1:1000, cell signaling), mouse anti-β-actin (1:5000, Millipore, Billerica, MA) as a control in a solution containing 0.05% Tween-20, 1% bovine serum albumin, and 4% nonfat dry milk at 4 °C overnight.

Techniques: Over Expression, Western Blot, Isolation