Total protein (50 μg) was separated in a Bis-Trispolyacryl amide gel and transferred onto a PVDF membrane (Millipore). The membrane was then incubated in 5% bovine serum albumin (BSA), and then incubated with a primaryantibody at 4°C overnight. Next, samples were incubated with IRDye800CWHRP-conjugated anti-IgG at room temperature for 1hour and visualized via chemiluminescence with an infrared laser scanning system (OdysseyLicor,Lincoln,NE,USA). The following primary rabbit-anti-human antibodies were used: anti-ANGPT2 (1:1000; Abcam); anti-Notch1 (1:500; Abcam); anti-Notch2 (1:500; Abcam); anti-Runx2 (1:1000;Abcam); anti-Sp7/Osterix (1:2000; Abcam); anti-ALP (1:2000; Abcam); anti-OCN (1:500; Abcam) and anti-GAPDH (1:2,500; Abcam).
Anti notch2
Manufactured by Abcam
Sourced in United States
Anti-Notch2 is a primary antibody that recognizes the Notch2 protein. Notch2 is a transmembrane receptor that plays a role in cell-cell signaling pathways. This antibody can be used in various research applications to detect and study the Notch2 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti notch1, by Abcam (3 mentions)
Anti notch3, by Abcam (2 mentions)
Anti ocn, by Abcam (1 mentions)
Anti runx2, by Abcam (1 mentions)
Anti angpt2, by Abcam (1 mentions)
Anti sp7 osterix, by Abcam (1 mentions)
Anti alp, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti phospho sapk jnk, by Cell Signaling Technology (1 mentions)
Anti sox2, by Abcam (1 mentions)
Anti jnk1 2, by Cell Signaling Technology (1 mentions)
Anti oct3 4, by Santa Cruz Biotechnology (1 mentions)
Anti phospho jnk, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti cd44, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
5 protocols using anti notch2
1
Western Blot Analysis of Cell Signaling Proteins
2
Molecular Profiling of Stem Cell Signaling
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Anti-CD44, anti-phospho-AKT, anti-AKT, anti-phospho-ERK, anti-ERK1/2, anti-phospho-p38, anti-p38, anti-phospho-SAPK/JNK, and anti-JNK1/2 antibodies were purchased from Cell Signaling Technology (Cambridge, MA, USA). Anti-phospho-JNK, anti-Oct3/4 and anti-β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-SOX2, anti-Notch2, and anti-Snail were purchased from Abcam (Cambridge, MA, USA). Anti-β-catenin was procured from BD Bioscience (Franklin Lakes, NJ, USA). For transfection, non-targeting siRNA and commercial siRNA for SOX2 or Snail or AKT were purchased from Genolution (Genolution Pharmaceuticals, Seoul, Korea). The cells were transfected with each siRNA (50 nM) for 48 h using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), as described in the manufacturer’s procedure.
3
Western Blot Analysis of Notch Signaling
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The protein samples were analyzed using a BCA kit (Pierce Company). The samples were separated by 10–20% Ready Gels (Bio-Rad, USA) and transferred onto a polyvinylidene fluoride (PVDF) membrane for 30 min in a transfer electricity meter (Bio-Rad, USA). The PVDF nitrocellulose films were cleared and sealed with 5% milk. After washing, anti-Notch 1 (1: 500, Abcam, USA), anti-Notch 2 (1: 800, Abcam, USA), anti-Notch 3 (1: 1000, Abcam, USA), and anti-DLL4 (1: 1000, Abcam, USA) were added to incubate overnight at 4°C. Then, these films were washed 3 times with Tris-buffered saline tween (TBST) and incubated with HRP secondary antibodies (1: 5000, Sigma, USA) at room temperature for 1 h. Relative expression levels of each protein were normalized to endogenous control β-actin using Image J software.
4
Notch Pathway Expression Analysis
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Immunohistochemistry was conducted by Tissue microarray analysis (TMA) of 76 specimens as previously described (20 (link)) following evaluation of tumor content and quality in hematoxylin and eosin-stained sections. TMAs were processed in a Leica BOND-III (Leica Biosystems, Nussloch, Germany) automated, continuous random, access slide-staining system that simultaneously processes multiple immunohistochemistry (IHC) assays. Protocol F was selected, with 3 min of heat-induced epitope retrieval and Bond Polymer Refine Detection (Leica Biosystems, DS9800) of primary antibodies. Anti-Notch1, anti-Notch2, anti-Notch3, anti-Notch4, anti-DLL1, anti-DLL2, anti-DLL3, and anti-SSTR2/5 (Abcam, Cambridge, UK) were the primary antibodies. Expression was assayed in photographs taken with an Aperio AT2 whole slide scanning system (Leica Biosystems). Staining intensity was scored as 0, no staining; 1, weak; 2, moderate; and 3, strong staining. An H-score was calculated from the percentage of positively stained cells at each intensity level using the following formula: [1 × (% weakly stained cells) + 2 × (% moderately stained) + 3 × (% strongly stained cells)].
5
Western Blot Analysis of MK and Notch2
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The total protein of ASMC cells from each group was extracted and prepared according to standard procedures and quanti ed using the BCA method. Proteins were separated on a 12% SDS polyacrylamide gel and transferred to a PVDF membrane. The membrane was incubated with primary antibodies (anti-MK, anti-Notch2, and anti-β-actin) (Abcam) at 4 ℃ overnight and then incubated with goat anti-rabbit IgG at room temperature for 1 h. The membrane was washed three times with TBST. Immunoreactive bands were visualized using the Tanon 5200 enhanced chemiluminescence detection system (Tanon, China) and quanti ed by densitometry using ImageJ. The relative expression of MK and Notch2 was measured in the four study groups.
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