Western blotting was conducted as described previously.[1 ] Briefly, an equal amount of protein extracts (30 µg) were subjected to SDS‐PAGE (15% gel for NPY and 12% gel for other antibodies) and transferred to PVDF membranes, followed by blocking for 1 h in 5% milk and incubation overnight at 4 °C with anti‐NPY (1:1000; Santa Cruz Biotechnology, Santa Cruz, USA), anti‐NPY1R (1:1000; Zen Bioscience, Chengdu, China), anti‐TEAD1 (1:1000; Servicebio), anti‐JUNB (1:1000; Zen Bioscience), anti‐CREB (1:1000; Servicebio), anti‐p‐CREB (1:1000; Zen Bioscience), or anti‐β‐actin (1:1000; Servicebio). After that, the membranes were washed and incubated at room temperature for 1 h with the secondary antibodies (1:5000; Servicebio). After washing, protein bands were examined by an enhanced chemiluminescence kit from Advansta (Menlo Park, USA).
Anti npy
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-NPY is a laboratory product offered by Santa Cruz Biotechnology. It is an antibody that specifically binds to the Neuropeptide Y (NPY) protein. NPY is a neurotransmitter found in the central and peripheral nervous systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Wuhan Servicebio Technology (1 mentions)
Enhanced chemiluminescence kit, by Advansta (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
Bovine insulin, by Merck Group (1 mentions)
2 deoxy d glucose, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Anti gsk3β, by Santa Cruz Biotechnology (1 mentions)
L 152 804, by Bio-Techne (1 mentions)
Bibp3226, by Bachem (1 mentions)
Anti pgsk3βser9, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Anti pomc, by Phoenix Pharmaceuticals (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Biosesang (1 mentions)
3 protocols using anti npy
1
Protein Expression Analysis by Western Blotting
2
Investigating NPY-Mediated Signaling Pathways
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Dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), bovine insulin, human NPY, and 2-deoxy-D-glucose were purchased from Sigma-Aldrich (St Louis, MO, USA). The Y5 receptor antagonist L-152,804 was purchased from Tocris Bioscience (Bristol, UK). The Y1 receptor antagonist BIBP-3226 (Diphenylacetyl-D-Arg-4-hydroxybenzylamide) was purchased from Bachem (San Carlos, CA, USA). 2-deoxy-D-[3H] glucose (2-[3H] DG) was obtained from Amersham Life Sciences (Buckinghamshire, UK).
Antibodies for immunoblot and immunofluorescence assays included: anti-NPY, anti-GSK3α, anti-GSK3β, anti-pGSK3αSer21, anti-pGSK3βSer9 (Santa Cruz Biotechnology, CA, USA); anti-PI3K, anti-PI3K p85, AKT, anti-pAKTSer473 (Cell Signaling Technology, MA, USA). Secondary antibodies conjugated to HRP and Alexa Fluor dyes for immunoblotting and immunofluorescence were purchased from Life Technologies (Grand Island, NY, USA). Antibodies were used according to manufacturer's instructions.
Antibodies for immunoblot and immunofluorescence assays included: anti-NPY, anti-GSK3α, anti-GSK3β, anti-pGSK3αSer21, anti-pGSK3βSer9 (Santa Cruz Biotechnology, CA, USA); anti-PI3K, anti-PI3K p85, AKT, anti-pAKTSer473 (Cell Signaling Technology, MA, USA). Secondary antibodies conjugated to HRP and Alexa Fluor dyes for immunoblotting and immunofluorescence were purchased from Life Technologies (Grand Island, NY, USA). Antibodies were used according to manufacturer's instructions.
3
Protein Expression in Hypothalamic Tissues
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The hypothalamus tissues of 3-week-old offspring (n=8) were homogenized in RIPA buffer (Biosesang, Seongnam, Korea) with a protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentration was determined using a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, USA). Forty micrograms of protein were separated on an 8% sodium dodecyl sulfate-polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was blocked for 1 hour in 5% skim milk in Tris-buffered saline with 0.01% tween-20 (TBS-T). After being washed, the membrane was incubated at 4°C overnight with an anti-POMC (1:1,000; Phoenix, Belmont, CA, USA), anti-NPY (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MC4R (1:1,000; Abcam, Cambridge, UK), or anti-beta-actin (1:3,000; Santa Cruz Biotechnology) antibody. The blot was then probed with the corresponding secondary antibody. The bands were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and densitometric analysis using ImageQuant software.
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