Quantitative evaluation of signaling events in Drosophila S2 cells

Bond, David; Primrose, David A.; Foley, Edan

doi:10.1251/bpo139

Cited by 19 publications

(17 citation statements)

References 25 publications

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“…Lysates were spun for 10 min at 16,000 rcf and dithiothreitol (DTT, Sigma-Aldrich) was added to the supernatants to reach a final concentration of 10 mM. Proteins were separated on a 9% SDS-PAGE gel and analyzed by Western blot [37]. The blots were incubated with rabbit anti-HA tag monoclonal antibody (clone C29F4, Cell Signaling, Danvers, MA), mouse anti-NELL1 monoclonal antibody (clone 6A8) and mouse anti-β-actin monoclonal antibody (clone AC-74, Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Effect of NELL1 gene overexpression in iPSC-MSCs seeded on calcium phosphate cement

et al. 2014

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Human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) are a promising source of patient-specific stem cells with great regenerative potential. There has been no report on NEL-like protein 1 (NELL1) gene modification of iPSC-MSCs. The objectives of this study were to genetically modify iPSC-MSCs with NELL1 overexpression for bone tissue engineering, and investigate the osteogenic differentiation of NELL1 gene-modified iPSC-MSCs seeded on Arg-Gly-Asp (RGD)-grafted calcium phosphate cement (CPC) scaffold. Cells were transduced with red fluorescence protein (RFP-iPSC-MSCs) or NELL1 (NELL1-iPSC-MSCs) by a lentiviral vector. Cell proliferation on RGD-grafted CPC scaffold, osteogenic differentiation and bone mineral synthesis were evaluated. RFP-iPSC-MSCs stably expressed high levels of RFP. Both the NELL1 gene and NELL1 protein levels were confirmed higher in NELL1-iPSC-MSCs than in RFP-iPSC-MSCs using RT-PCR and Western blot (p < 0.05). Alkaline phosphatase (ALP) activity was increased by 130% by NELL1 overexpression at 14 d (p < 0.05), indicating that NELL1 promoted iPSC-MSC osteogenic differentiation. When seeded on RGD-grafted CPC, NELL1-iPSC-MSCs attached and expanded similarly well to RFP-iPSC-MSCs. At 14 d, runt-related transcription factor 2 (RUNX2) gene level of NELL1-iPSC-MSCs was 2.0-fold that of RFP-iPSC-MSCs. Osteocalcin (OC) level of NELL1-iPSC-MSCs was 3.1-fold that of RFP-iPSC-MSCs (p < 0.05). Collagen type I alpha 1 (COL1A1) gene level of NELL1-iPSC-MSCs was 1.7-fold that of RFP-iPSC-MSCs at 7 d (p < 0.05). Mineral synthesis was increased by 81% in NELL1-iPSC-MSCs at 21 d. In conclusion, NELL1 overexpression greatly enhanced the osteogenic differentiation and mineral synthesis of iPSC-MSCs on RGD-grafted CPC scaffold for the first time. The novel NELL1-iPSC-MSC seeded RGD-CPC construct is promising to enhance bone engineering.

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Section: Methodsmentioning

confidence: 99%

Effect of NELL1 gene overexpression in iPSC-MSCs seeded on calcium phosphate cement

et al. 2014

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“…Membranes were then washed three times for 5 min each in 0.01% Tween phosphate buffered saline, and then briefly washed in water three times. Blots were imaged with the LiCor Odyssey laser based imaging system (Bond et al, 2008). …”

Section: Methodsmentioning

confidence: 99%

“…Using the Odyssey 3.0 analytical software, near infrared fluorescent signals obtained from the LiCor Odyssey scanner were expressed as raw integrated intensity with top–bottom median intra-lane background subtraction (Bond et al, 2008). Samples were run in duplicate.…”

Section: Methodsmentioning

confidence: 99%

Regional differences in expression of β-tubulin isoforms in schizophrenia

Moehle

Ludueña

Haroutunian

et al. 2012

Schizophrenia Research

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A growing body of evidence suggests that abnormal elements of the cytoskeleton may be associated with the pathophysiology of schizophrenia. Isoforms of a major cytoskeleton protein, β-tubulin, were recently demonstrated to have distinct roles in neuronal differentiation and cell viability. For these reasons, we tested the hypothesis that there are differences in the expression of β-tubulin isoforms (βI-βIV) in the brain in schizophrenia, using western blot analysis in an elderly group of subjects with this illness and a control group. We found that βI-tubulin protein expression was decreased in the anterior cingulate cortex and increased in the dorsolateral prefrontal cortex, but not changed in superior temporal gyrus or hippocampus in schizophrenia. Our data supports the growing body of evidence suggesting abnormalities of the cytoskeleton in schizophrenia.

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“…Membranes were washed again with 0.01% Tween phosphate buffered saline four times for 5 minutes each and then briefly rinsed 3 times in distilled water. The blots were stored in distilled water at 4° C until scanned using the LI-COR Odyssey® laser-based image detection method (Bond et al , 2008). We tested each antibody using varying concentrations of total protein from homogenized human cortical tissue to confirm we were in the linear range of the assay.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Abnormal Forward Trafficking of AMPA Receptors in Frontal Cortex of Elderly Patients with Schizophrenia

Hammond

McCullumsmith

Funk

et al. 2010

Neuropsychopharmacol

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Several lines of evidence point to alterations of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor trafficking in schizophrenia. Multiple proteins, including Synapse Associated Protein 97 (SAP97), Glutamate Receptor Interacting Protein 1 (GRIP1), and N-ethylmaleimide Sensitive Factor (NSF), facilitate the forward trafficking of AMPA receptors toward the synapse. Once localized to the synapse, AMPA receptors are trafficked in a complex endosomal system. We hypothesized that alterations in the expression of these proteins and alterations in the subcellular localization of AMPA receptors in endosomes may contribute to the pathophysiology of schizophrenia. Accordingly, we measured protein expression of SAP97, GRIP1, and NSF in the dorsolateral prefrontal cortex and found an increase in the expression of SAP97 and GRIP1 in schizophrenia. To determine the subcellular localization of AMPA receptor subunits, we developed a technique to isolate early endosomes from postmortem tissue. We found increased GluR1 receptor subunit protein in early endosomes in subjects with schizophrenia. Together, these data suggest that there is an alteration of forward trafficking of AMPA receptors as well as changes in the subcellular localization of an AMPA receptor subunit in schizophrenia.

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Quantitative evaluation of signaling events in Drosophila S2 cells

Cited by 19 publications

References 25 publications

Effect of NELL1 gene overexpression in iPSC-MSCs seeded on calcium phosphate cement

Effect of NELL1 gene overexpression in iPSC-MSCs seeded on calcium phosphate cement

Regional differences in expression of β-tubulin isoforms in schizophrenia

Evidence for Abnormal Forward Trafficking of AMPA Receptors in Frontal Cortex of Elderly Patients with Schizophrenia

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