Whole cell lysates were prepared as described 13 (link), 36 (link). 20 µg of denatured protein was fractionated on a NuPAGE Bis-Tris 4–12% gel (Invitrogen). Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser780; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.
Anti jagged1 28h8
Manufactured by Cell Signaling Technology
Anti-Jagged1 28H8 is a lab equipment product manufactured by Cell Signaling Technology. It is a monoclonal antibody that recognizes the Jagged1 protein, a type I transmembrane protein that functions as a ligand for the Notch receptor.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated secondary antibody, by GE Healthcare (2 mentions)
Anti phospho rb ser780, by Cell Signaling Technology (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Icn1val1744, by Cell Signaling Technology (2 mentions)
Notch1 val1744 d3b8, by Cell Signaling Technology (2 mentions)
Anti notch3 8g5, by Cell Signaling Technology (2 mentions)
Anti human p16 g175 1239, by BD (2 mentions)
Anti p15 15p06, by Santa Cruz Biotechnology (2 mentions)
Anti involucrin clone sy5, by Merck Group (2 mentions)
Anti β actin ac 74, by Merck Group (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Anti actin, by Merck Group (1 mentions)
3 protocols using anti jagged1 28h8
1
Western Blot Analysis of Notch Signaling
2
Western Blot Analysis of Notch Signaling
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Whole cell lysates were prepared as described 13 (link), 36 (link). 20 µg of denatured protein was fractionated on a NuPAGE Bis-Tris 4–12% gel (Invitrogen). Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser780; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.
3
Characterization of GnRH-secreting Neuroblasts
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HEK293T cells (sourced from ATCC) and GN11 cells (56 (link)) (gift of Sally Radovick, Rutgers University, New Brunswick, New Jersey, USA) were grown in monolayer at 37°C under 5% CO2 in DMEM (Thermo Fisher Scientific, 11965092) containing 100 μg/mL streptomycin, 100 U/mL penicillin (Thermo Fisher Scientific, 15140122), supplemented with 10% FBS (Thermo Fisher Scientific, 26140079). FNCB4 cells were previously established, cloned, and propagated in vitro from the human fetal OE (27 (link)) and characterized as migratory GnRH-secreting neuroblasts (28 (link)). Cells, cryogenically preserved, were cultured at 37°C in 5% CO2 atmosphere in Coon’s modified Ham F-12 medium (MilliporeSigma, F6636) supplemented with 10% FBS. Cells were maintained below full confluence by trypsinization and seeding onto 10 cm² dishes. HEK293T cells were seeded (2.5 × 105 cell/well) on 6-well plates and transfected 24 hours later with Lipofectamine 3000 Reagent (Thermo Fisher Scientific, L3000015) according to the data sheet, using 1 μg of plasmid.
Western blot experiments were carried out on HEK293T cell lysates using antibodies anti-Jagged1 (28H8) (Cell Signaling Technology, 2620) and anti-Actin 1:5,000 (MilliporeSigma, A5060) as internal control.
Western blot experiments were carried out on HEK293T cell lysates using antibodies anti-Jagged1 (28H8) (Cell Signaling Technology, 2620) and anti-Actin 1:5,000 (MilliporeSigma, A5060) as internal control.
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