Proteins were separated by SDS-PAGE and transferred to a Protran nitrocellulose membrane (GE Healthcare Life Sciences) using a wet transfer apparatus (Amersham Bioscience). The membranes were blocked for unspecific binding with 5% nonfat dry milk at RT for 1 hour. Primary antibody incubation was performed for 1 hour at RT, followed by a secondary antibody incubation for 1 hour at RT (1 : 4000). Proteins were detected using SuperSignal West Pico PLUS Enhanced chemiluminescence substrate (ThermoFisher) following the manufacturer's instructions. The following antibodies were used for western blot: β-actin (Cell Signaling Technology), Notch1 (C20, Santa Cruz), Jagged1 (28H8, Cell Signaling Technology), both 1 : 1000 dilution.
Jagged1 28h8
Manufactured by Cell Signaling Technology
Jagged1 (28H8) is a mouse monoclonal antibody that recognizes the Jagged1 protein. Jagged1 is a ligand for the Notch receptor family and plays a role in cell-cell signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Protran nitrocellulose membrane, by GE Healthcare (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Notch1 c 20, by Santa Cruz Biotechnology (1 mentions)
Protran, by Cytiva (1 mentions)
Facscalibur, by BD (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Cd34 apc, by BD (1 mentions)
Streptavidin pe cy7, by BD (1 mentions)
Cd271 apc, by BD (1 mentions)
Cd146 pecy7, by BD (1 mentions)
Cd140a pe, by BD (1 mentions)
2 protocols using jagged1 28h8
1
Western Blot Analysis of Notch Signaling
2
Multiparametric Flow Cytometry Analysis of MSCs
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Flow cytometry analysis of MSC surface markers was performed as described [42 (link)]. Briefly, cells were stained with monoclonal antibodies, anti-human CD73-PE, CD-34-APC, CD146-PEcy7, CD271-APC, Streptavidin-PEcy7, CD140a-PE (BD Pharmingen), SSEA4-Biotin (R&D Systems, Minneapolis, MN), CD166-FITC (Serotec, Oxford, UK) and analyzed using FACSCalibur (Becton Dickinson) and CellQuest software. To examine the expression of cross-talk molecules, MSCs were permeabilized and intracellular stained with specific antibodies against Jagged-1(28H8, Cell signaling, Danvers, MA) or CXCL-12 (79018, R&D Systems) as described [13 (link), 43 ]. Relative expression levels were determined by ΔMFI, difference in mena fluorescent intensity. Osteogenic differentiation and adipogenic differentiation of MSCs were induced by each specific differentiation medium and quantified by Alizarin Red staining or lipid droplet as previously described [19 (link)]. For colony formation (CFU-F), MSCs were plated at a density of 500 cells per 100 mm dish, and after incubation for 14 days, the number of colonies containing >50 cells was counted after staining with crystal violet (Sigma) in methanol.
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