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 involucrin clone sy5
Manufactured by Merck Group
Sourced in United States
Anti-Involucrin clone SY5 is a laboratory reagent used to detect the presence of the involucrin protein in biological samples. Involucrin is a protein involved in the terminal differentiation of keratinocytes, which are the predominant cell type in the outermost layer of the skin. The Anti-Involucrin clone SY5 can be used in various laboratory techniques, such as immunohistochemistry, to identify and analyze the distribution of involucrin in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated secondary antibody, by GE Healthcare (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Anti jagged1 28h8, by Cell Signaling Technology (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)
β actin, by Merck Group (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Anti phospho rb ser780, by Cell Signaling Technology (2 mentions)
Anti β actin ac 74, by Merck Group (2 mentions)
Alexa 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti collagen 1, by Abcam (1 mentions)
Anti collagen 4, by Abcam (1 mentions)
Anti fibronectin, by Abcam (1 mentions)
Anti vimentin clone v9, by Agilent Technologies (1 mentions)
Anti α smooth muscle actin clone 1a4, by Merck Group (1 mentions)
Anti thy1 clone 5e10, by BD (1 mentions)
Anti ki 67, by Roche (1 mentions)
4 protocols using anti involucrin clone sy5
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
Antibody Characterization for Cell Phenotyping
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The primary rabbit polyclonal antibodies used were: anti-collagen I (Abcam; Cambridge, UK), anti-collagen IV (Abcam), anti-fibronectin (Abcam) and the anti-perlecan antibody CCN-1 was a gift from Prof. John Whitelock (University of NSW, Sydney, Australia). The mouse monoclonal antibodies (mAbs) used were: anti-fibroblast marker (clone TE7; Millipore; MA, USA), anti-involucrin (clone SY5; Sigma; MO, USA), anti-ki67 (clone MM1; Novacastra; Wetzlar, Germant), anti-p63 (clone 4A4; Abcam), anti-α-smooth muscle actin (clone 1A4; Sigma), anti-Thy1 (clone 5E10, BD Bioscience; NJ, USA) and anti-vimentin (clone V9; Dako; CA, USA). The mouse mAbs anti-keratin 14 (K14, clone LL001), anti-keratin 16 (K16, clone LL025) and anti-keratin 10 (K10, clone LH2) were produced in the labs of EBL and IM Leigh71 (link),72 (link). The secondary antibodies used were Alexa488 anti-mouse IgG, Alexa546 anti-mouse IgG, Alexa 488 anti-rabbit IgG and Alexa546 anti-rabbit IgG (all from Molecular Probes, ThermoFisher Scientific; OR, USA).
4
Antibody-based Analysis of Cellular Signaling
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An anti-human APC-CD271 antibody (clone ME20.4-1.H4, Miltenyi Biotec, Germany) was used for flow cytometry analysis. The following antibodies were used for Western blotting analysis: anti-phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204) (20G11, Cell Signaling Technologies, Danvers, MA, USA), anti-ERK (137E5, Cell Signaling), anti-phospho-NF-κB p65 (S536) and anti-NF-κB p65 (93H1 and D14E12, Cell Signaling), anti-phospho-Iκbα (Ser32) and anti-Iκbα (14D4 and L35A5, Cell Signaling), anti-phospho- Akt (S473) and anti-Akt (pan, Cell Signaling) anti-α-tubulin (B-5-1-2, Santa Cruz Biotechnology, Dallas, TX, USA), anti-involucrin (clone SY-5, Sigma-Aldrich, St. Louis, IL, USA), anti-CDKN1C (NBP1-89917, Novus Biologicals, Littleton, CO, USA), and anti-β-actin (AC-15, Sigma-Aldrich). The following antibodies were used for immunohistochemistry analysis: anti-CD271 (C40-1457, BD Biosciences, Franklin Lakes, NJ, USA), anti-Ki67 [clone 30-9 (Roche, Switzerland) for DAB staining and clone SP6 (Abcam, UK) for immunofluorescence staining] and anti-involucrin (clone SY-5, Sigma-Aldrich).
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