Journal: The Journal of Neuroscience

Article Title: CNS Progenitor Cells Promote a Permissive Environment for Neurite Outgrowth via a Matrix Metalloproteinase-2-Dependent Mechanism

doi: 10.1523/JNEUROSCI.0200-07.2007

Figure Lengend Snippet: Degradation of CD44 and neurocan by MMP2 in vitro. A–D, Incubation of rd1 retinas with recombinant MMP2 for 12 h led to a decrease in CD44 (A) and neurocan (B) immunoreactivity on the outer surface of rd1 retinas (arrowheads) compared with controls. However, the treatment did not appreciably change GFAP immunoreactivity in reactive Müller cells (C) or significantly alter retinal morphology (D). E, F, Western blot shows CD44 (E) and neurocan (F) cleavage products in rd1 explants treated with conditioned medium from rd1 retina/RPC cocultures. Lanes 1–3, Protein was extracted in culture medium from rd1 explants treated with conditioned medium from RPC (lane 1), rd1 retina/RPC cocultures (lane 2), or rd1 retina/NPC cocultures (lane 3). Lanes 4, 5, Protein was extracted from RPC (lane 4) or NPC lysate (lane 5). Scale bars: A–C, 30 μm; D, 200 μm.

Article Snippet: The primary antibodies used were raised against GFP (1:100; Sigma), CD44 (1:100; Sigma), neurocan (1:500; Renee K. Margolis, Department of Pharmacology, State University of New York, Brooklyn, NY), MMP2 (1:50; Chemicon, Temecula, CA), MMP14 (1:100; Chemicon), PKC (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), nestin (1:1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), neurofilament 200 (1:1000; Sigma), calbindin (D-28K, 1:800; Sigma), calretinin (1:100; Chemicon), rhodopsin (2D4, 1:500; gift from Dr. Robert Molday, University of British Columbia, Vancouver, British Columbia, Canada; 1:500), recoverin (1:200; Chemicon), cellular retinaldehyde-binding protein (1:500; Chemicon), and GFAP (1:400; Invitrogen).

Techniques: In Vitro, Incubation, Recombinant, Western Blot

Journal: The Journal of Neuroscience

Article Title: CNS Progenitor Cells Promote a Permissive Environment for Neurite Outgrowth via a Matrix Metalloproteinase-2-Dependent Mechanism

doi: 10.1523/JNEUROSCI.0200-07.2007

Figure Lengend Snippet: Reduction of CD44 and neurocan in rd1 retinas after engraftment with progenitor cells in vivo. CD44 (A) and neurocan (B) immunoreactivity was greatly reduced (arrowheads in left column) along the interface of graft and host in regions in which RPCs had migrated into the host retina at 30 d after transplantation (left column) compared with controls in which rd1 retinas were engrafted with neural retinal tissue (NR-rd1; right column). Scale bars, 30 μm.

Article Snippet: The primary antibodies used were raised against GFP (1:100; Sigma), CD44 (1:100; Sigma), neurocan (1:500; Renee K. Margolis, Department of Pharmacology, State University of New York, Brooklyn, NY), MMP2 (1:50; Chemicon, Temecula, CA), MMP14 (1:100; Chemicon), PKC (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), nestin (1:1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), neurofilament 200 (1:1000; Sigma), calbindin (D-28K, 1:800; Sigma), calretinin (1:100; Chemicon), rhodopsin (2D4, 1:500; gift from Dr. Robert Molday, University of British Columbia, Vancouver, British Columbia, Canada; 1:500), recoverin (1:200; Chemicon), cellular retinaldehyde-binding protein (1:500; Chemicon), and GFAP (1:400; Invitrogen).

Techniques: In Vivo, Transplantation Assay

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Differentially expressed genes and Kyoto Encyclopedia of Genes and Genomes analysis. Volcano plots showing the top twenty up- or downregulated genes for each cell type. The red dots represent upregulated genes, and the blue dots represent downregulated genes. ( P value < 0.05, and |log2foldchange| > 0.58). Bubble plot indicating the top enriched pathways for each cell type based on KEGGpathway enrichment analysis of differentially expressed genes( www.kegg.jp/kegg/kegg1.html ). The sizes of the dots represent the number of genes included in each pathway. The colour gradient of dots represents the adjusted P values of each enriched pathway. The genes Pfkfb3 , Tbc1d1 and the insulin signaling pathway; Pdgfd and the PI3K-Akt signaling pathway; Cxcr2 and PLD, Rap1 signaling pathways; Ccl21 and chemical carcinogenesis-reactive oxygen species signaling pathway; Mef2c and the calcium signaling pathway; Negr1 and leukocyte transendothelial migration signaling pathway are labelled by the black and red bars in Panels a , b , c , d , e , and f .

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Protein-Protein interactions, Migration

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Protein-protein interaction network (PPI network).We constructed nine PPI networks displaying protein-protein interactions among the related genes. The nodes represent proteins, and the edges represent the interaction strength between two proteins. The proteins PFKFB3 ( a ), PDGFD ( b ), CXCR2 ( c ), CCL21 ( d ), SEMA3A ( e ), RYR3 ( f ), MEF2C ( g ), NEGR1( h ) and TBC1D1 ( i ) which are located at the hub of the interaction network, are responsible for diaphragm fibrosis and atrophy.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Construct, Protein-Protein interactions

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR and Western blotting showing the expression of selected genes and corresponding proteins in the mechanically ventilated diaphragm and control ones. The bar graphs show the quantification of the mRNA expression of Pfkfb3 ( a ), Pdgfd ( b ), Cxcr2 ( c ), Negr1 ( d ), Sema3a ( e ), and Mef2c ( f ) normalized to that of GAPDH. Asterisks indicate significant differences ( n = 3 in each group) (* P < 0.05). Western blotting analysis of the protein expression of PFKFB3 ( g ), PDGFD ( h ), CXCR2 ( i ), NEGR1 ( j ), SEMA3A ( k ), and MEF2C ( l ) normalized to that of β-actin as a loading control.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: Molecular Neurobiology

Article Title: Runx2 Suppresses Astrocyte Activation and Astroglial Scar Formation After Spinal Cord Injury in Mice

doi: 10.1007/s12035-024-04212-6

Figure Lengend Snippet: Overexpressing Runx2 inhibited the activation of astrocytes following SCI in vivo. A Western blot analysis of Neurocan (143 kDa) and GFAP (55 kDa)in lesions, with GAPDH (37 kDa) as an internal loading control. B , C Quantification of result in panel ( A ). The data were analyzed using one-way analysis of variance and all data are expressed as the mean ± S.D. D Immunofluorescence staining of DAPI (blue), GFAP (red), and Runx2 (green) in the spinal cord at 14 days post-injury. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar = 200 μm. Runx2-OE (AAV-Runx2)

Article Snippet: Then incubate overnight at 4 °C with the following primary antibodies: Runx2 (1:1000; Abcam); GFAP (1:1000; Abcam); polyclonal GAPDH antibody (1:1000; Proteintech); polyclonal S100A10 antibody (1:1000; Proteintech); Neurocan (1:1000; Zenbio); and NF-κB p65 (1:1000; Proteintech).

Techniques: Activation Assay, In Vivo, Western Blot, Control, Immunofluorescence, Staining

Journal: Molecular Neurobiology

Article Title: Runx2 Suppresses Astrocyte Activation and Astroglial Scar Formation After Spinal Cord Injury in Mice

doi: 10.1007/s12035-024-04212-6

Figure Lengend Snippet: The effect of Runx2 nuclear import is crucial on modulation of astroglial scar. A Western blot analysis of Runx2 in nucleus (57 kDa) and GFAP (55 kDa), with GAPDH (37 kDa) as an internal loading control. B , C Quantification of result in panel ( A ). D Immunofluorescence staining of DAPI (blue), GFAP (red), and Runx2 (green) in the SVG-P12. E Immunofluorescence staining of DAPI (blue), Neurocan (red), and Runx2 (green) in the SVG-P12. The data were analyzed using one‑way analysis of variance, and all data are expressed as the mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar = 20 μm. DC (plasmid without Runx2), DR (plasmid of Runx2 with NMTS), DM (plasmid of Runx2 without NMTS)

Article Snippet: Then incubate overnight at 4 °C with the following primary antibodies: Runx2 (1:1000; Abcam); GFAP (1:1000; Abcam); polyclonal GAPDH antibody (1:1000; Proteintech); polyclonal S100A10 antibody (1:1000; Proteintech); Neurocan (1:1000; Zenbio); and NF-κB p65 (1:1000; Proteintech).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Development of Neurogenic Detrusor Overactivity after Thoracic Spinal Cord Injury Is Accompanied by Time-Dependent Changes in Lumbosacral Expression of Axonal Growth Regulators

doi: 10.3390/ijms23158667

Figure Lengend Snippet: Primary antibodies used in Western Blotting, Immunohistochemistry and Immunocitochemistry assays.

Article Snippet: Neurocan , MAB5212 , Merck Millipore , 245, 160, 130 and 90 kDa , Mouse , 1:1000 , 1:1000.

Techniques: Western Blot, Immunohistochemistry