Protein lysates in NuPAGE buffer (Invitrogen) were separated by gradient 4–12% BisTris Gel and transferred to polyvinylidene difluoride membrane. Primary antibodies used in these experiments included rabbit monoclonal anti-Notch1 (D1E11; 1:1000; Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-cleaved Notch1 (Val1744; 1:1000; Cell Signaling), rabbit monoclonal anti-Notch2 (8A1; 1:1000; Cell Signaling), rabbit polyclonal anti-Notch3 (Pro2311; 1:1000; Cell Signaling), rabbit polyclonal anti-HES1 (ab71559; 1:1000; Abcam, Cambridge, MA, USA), rabbit polyclonal anti-HEY1 (ab22614; 1:500; Abcam), rabbit polyclonal anti-HES6 (ab66461; 1:1000; Abcam) and mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (clone 6C5; 1:1000; Santa Cruz Biotechnology, Dallas, Tx, USA). The membrane was incubated sequentially with primary antibodies (overnight at 4°C), horseradish peroxidase-conjugated anti-rabbit or antimouse secondary antibodies, and chemiluminescent substrate (Thermo Fisher, Waltham, MA, USA) before exposure to film.
Ab71559
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab71559 is a primary antibody produced in rabbit. It is designed for use in immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab52627, by Abcam (9 mentions)
Ab8925, by Abcam (6 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ab22614, by Abcam (2 mentions)
Gtx42614, by GeneTex (2 mentions)
Ab128076, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab76655, by Abcam (2 mentions)
Ab7771, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ab66461, by Abcam (1 mentions)
Val1744, by Cell Signaling Technology (1 mentions)
Anti ubiquitin, by Merck Group (1 mentions)
Ab23426, by Abcam (1 mentions)
Ab184742, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Par 4335 mc 100 ac, by Bio-Techne (1 mentions)
24 protocols using ab71559
1
Notch Signaling Pathway Protein Analysis
2
Investigating Notch Signaling Pathway
3
Immunostaining and Western Blot Analysis of Notch1 Signaling
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H&E and IF were performed as described [11 (link)]. Antibodies recognize activated Notch1, Hes1, Ki67, Luc (ab8925, ab71559, ab15580, ab81823, Abcam, Cambridge, MA), Hey-1, and FSP1 (GTX42614, GTX89197, GeneTex, Irvine, CA). Nuclei were stained with DAPI (Sigma-Aldrich, St. Louis, MO). Quantifications are mean ± standard deviation (SD) of counts from 5 low power field (LPF) per tumor sample for H&E staining, and 5 high power field (HPF) per section and 5 section/tumor for IF staining. For Western blot, skin tissue samples were homogenized in a RIPA buffer (50 mM Tris-Cl, 150 mM sodium chloride 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0, 1 mM EDTA and protease inhibitor cocktails (Roche)). The tissue suspension was rotated at 4°C for 30 min. Supernatants were collected after centrifugation at 13,000 rpm for 15 min. Concentration of protein was determined using PierceTM BCA Protein Assay Kit (#23225). Western blot was conducted as described [11 (link)]. Expression of mutant N1IC (muN1IC, 59Kd) and deletion of Notch1 in mouse skin were detected by two different anti-Notch1 antibodies, respectively (ab8925 and ab52627, Abcam).
4
Protein Expression Analysis of Notch Signaling Pathway
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Total protein was extracted using RIPA buffer (KeyGen, China). Protein concentration was quantified using a BCA protein kit (Beyotime, China). Proteins (30 µg) were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% milk, membranes were incubated with primary antibodies overnight at 4°C, including Numb (ab155415, Abcam, USA), Numbl (ab37500, Abcam, USA), Notch1 (ab128076, Abcam, USA), Hes1 (ab71559, Abcam, USA), and β-actin (ab8227, Abcam, USA). Subsequently, the samples were incubated with secondary antibodies (Abcam, USA) for 1.5 h at room temperature and analyzed using enhanced chemiluminescence reagent (Beyotime, China) [28 (link)].
5
Molecular Regulation of Notch Signaling
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TCP was purchased from MedChemExpress (USA). Primary antibodies recognizing LSD1 (ab17721) and Hes1 (ab71559) were obtained from Abcam (UK), DTX1 (GTX112367) were obtained from GeneTex (USA), and H3 (9728S), Notch 1 (3608S), Notch 3 (3889S), H3K4me2 (3889S), PI3K (4228S), Rictor (9476S), p-Akt (Ser473) (4060S), Akt (2920S), p-mTOR (Ser2448) (5536S), p-mTOR (Ser2481) (2974S), mTOR (2983S), Raptor (2280S), p-p70S6K (Thr389) (9206S) and GAPDH (5174S) as well as the secondary antibodies were obtained from Cell Signaling Technology (USA). LSD1 shRNA and control shRNA vector were obtained from GenePharma Company (Shanghai, China).
6
Notch Pathway Profiling by RT2-PCR
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The Human Notch Pathway RT2-Profiler™ PCR Array (#PAHS-059, Invitrogene) was conducted as described [9 (link)]. All samples were normalized to the levels of β-actin, and results are expressed as relative fluorescence intensity. 4%-20% gradient SDS-PAGE gel was used for Hes-1 in Fig 2C -related experiment, while 10% SDS-PAGE gels were used for other Western blot experiments. Immunoblot was performed as described [17 (link)]. Membranes were probed with Abs to Notch1 (ab52627, Abcam for Fig 2C ; ab27526, Abcam for Fig 2D ; and N4788, Sigma-Aldrich for Fig 3A , respectively), Notch4 (TA321658, Origene) that detected full length Notch4 expression in MAFs, activated Notch1 (for Fig 3A , N4788, Sigma-Aldrich), Hes-1 (ab71559, Abcam for Fig 2D and Fig 3A ; ab55265, Abcam for Fig 2C , respectively), Hey-1 (GTX42614, GeneTex), WISP-1 (SAB1400324, Sigma-Aldrich) or β-actin (A1978, Sigma-Aldrich) accordingly. Autophotographs of blots were scanned by densitometer (Molecular Dynamics) to quantify the bands.
7
Protein Quantification and Analysis of Sciatic Nerve Samples
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After removal of the perineurium, SNs were homogenized in microcentrifugation tubes (Eppendorf) using chilled pestles (Argos Technologies) in lysis buffer (2% SDS, 10 mM NaCl, 25 mM Tris-HCl pH 7.4, proteinase inhibitor cocktail (Sigma), phosphatase inhibitor (Roche)). Lysates were supplemented with sample buffer (6.2 mM Tris, 1% β-mercaptoethanol, 2% glycerol) and further processed using standard SDS-PAGE and western blot methods. The following antibodies were used: Notch1 (MAB5352, Millipore, 1:1,000), HES1 (ab71559, Abcam, 1:1,000), EGR2 (13491-1-AP, Proteintech, 1:1,000), E-cadherin (ALX-804-202-C100, Enzo, 1:1,000), HMGA2 (#8179, Cell Signalling, 1:1,000), β-actin (A5316, Sigma, 1:5,000), MBP (MCA409s, Serotec, 1:1,000). Secondary antibodies were obtained from Jackson and Promega. Signal detection was carried out using Fusion FX7 (Vilber Lourmat) and band intensities were quantified using Quantity One software (Biorad). Uncropped blots are shown in Supplementary Fig. 5 . Size markers refer to All Blue Precision Protein Standards (Biorad).
8
Immunohistochemistry Analysis of HES1 Expression
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Immunohistochemistry analysis was carried out according to the Envision System (Dako Cytomation, Glostrup, Denmark) guidance. In brief, each TMA slides was deparaffinzed and rehydrated through graded ethanol. Sodium citrate was used for antigen retrieval. Slides underwent 0.3% hydrogen peroxide solution to block endogenous peroxidase activity. Then samples was incubated with the primary antibody anti-HES1 (1:400; Abcam, ab71559), at 4°C overnight. After incubation with secondary (goat) antibody, slides were developed in diaminobenzine (EnVision, DAKO) and counterstained with haematoxylin.
9
Notch Signaling Pathway Protein Detection
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Samples were run by SDS-PAGE and transferred to low autofluorescence polyvinylidene difluoride membrane (Immobilon, FL, Millipore). Primary antibodies were used at the concentration described below overnight at 4 °C in blocking solution (1% milk powder in PBS). Secondary antibody (IR-Dye conjugation goat anti-mouse or -rabbit LI-COR Biosciences) was used at 1:50,000 dilution in blocking solution for 1 h. The membrane was scanned on the LI-COR Odyssey (LI-COR Biosciences) using the Image Studio software.
Dilution of primary antibodies. 1:250: Notch1 (ab52627, Abcam), NICD (ab8925, Abcam), Hes1(ab71559, Abcam), Dll1 (ab76655, Abcam), ATG7 (ab52472, Abcam), Numb (ab14140, Abcam)
1:1,000: LC3 (NB 100-2220, Novus), ATG16L1 (pAb PM040, MBL), Beclin (#3738S, Cell Signalling), VAMP3 (gift from A.A. Peden)
1:2,000: Actin (A2066,Sigma-Aldrich).
Notch1 on western blot shows Notch NTMD (125 kDa). It is the form which is cleaved during maturation at the plasma membrane. The NICD (activated Notch1) antibody detects VLLSRKRRRQHGQC, a sequence, which is not accessible in the uncleaved form. It is exposed after S1 cleavage. The protein is detected at 80 kDa.
Dilution of primary antibodies. 1:250: Notch1 (ab52627, Abcam), NICD (ab8925, Abcam), Hes1(ab71559, Abcam), Dll1 (ab76655, Abcam), ATG7 (ab52472, Abcam), Numb (ab14140, Abcam)
1:1,000: LC3 (NB 100-2220, Novus), ATG16L1 (pAb PM040, MBL), Beclin (#3738S, Cell Signalling), VAMP3 (gift from A.A. Peden)
1:2,000: Actin (A2066,Sigma-Aldrich).
Notch1 on western blot shows Notch NTMD (125 kDa). It is the form which is cleaved during maturation at the plasma membrane. The NICD (activated Notch1) antibody detects VLLSRKRRRQHGQC, a sequence, which is not accessible in the uncleaved form. It is exposed after S1 cleavage. The protein is detected at 80 kDa.
10
Proteomic Analysis of Notch Signaling
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Cell lysates were separated by SDS PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed by using primary antibodies against ASPH; JAG1, JAG2, DLL1, DLL4, Notch1, Notch2, Notch3, Notch4, c-Myc, matrix metalloproteinase (MMP)-2, epithelial cell adhesion molecule (EpCAM), and CD44 (Cell Signaling Technology #2620, #2205, #2588, #2589, #4380, #4530, #5276, #2423, #5605, #13132, #2929, #5640, respectively); activated Notch1, HES1 and HEY1 (Abcam ab8925, ab71559, ab22614, respectively); cyclin D3, MMP9, and proliferating cell nuclear antigen (PCNA) (Santa Cruz, sc-56307, sc-21733, sc-7907, respectively). Protein bands were visualized by IRDye® 680RD Infrared Dye and IRDye® 800CW Infrared Dye and exposed on Odyssey image system (LI-COR).
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