Cells were plated on glass coverslips (Fisher Scientific) in 12-well culture dishes and grown to approximately 50% confluence for 24 h to promote adherence. The cells were then fixed with 4% paraformaldehyde for 10 min and permeabilised with PBS containing 0.1% Triton X-100 for 15 min. They were then incubated with blocking solution (Beyotime) for 30 min. Samples were incubated with primary antibody (α-SMA and NPY(1–36), Sigma; NPY1R, AbCam) at 4 °C overnight and then incubated with Alexa Fluor 488 or Alexa Fluor 555 secondary antibody (Invitrogen) at room temperature for 1 h. Nuclei were stained with Hoechst 33342 (Invitrogen) for 5 min. Following a final three washes with PBS, the cells were imaged by fluorescence microscopy DMI4000B (Leica).
Npy1r
Manufactured by Abcam
Sourced in United States
NPY1R is a protein-coding gene that produces the neuropeptide Y receptor type 1 protein. This receptor is involved in the regulation of various physiological processes, including food intake, energy homeostasis, and the stress response.
Automatically generated - may contain errors
Lab products found in correlation
Blocking solution, by Beyotime (1 mentions)
Glass coverslip, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Dmi4000b, by Leica camera (1 mentions)
Enhanced chemiluminescence, by Beyotime (1 mentions)
Runx2, by Bioss Antibodies (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Gapdh, by Keygen Biotech (1 mentions)
Ccnd1, by Cell Signaling Technology (1 mentions)
Rna polymerase 2 pol 2, by Santa Cruz Biotechnology (1 mentions)
Rabbit igg, by GE Healthcare (1 mentions)
Acetyl histone h3, by Abcam (1 mentions)
Cleaved parp 1, by Cell Signaling Technology (1 mentions)
Mouse or rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Tri methyl histone h3, by Cell Signaling Technology (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
3 protocols using npy1r
1
Immunostaining of Cytoskeletal and Neuropeptide Markers
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted from bone using RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1% phenylmethylsulphonyl fluoride. A bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology) was used to determine the protein concentration. The sample was resolved using 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and blocked with 5% skim milk. The membrane was incubated with primary antibodies against Zip1 (1: 100; Santa Cruz Biotechnology, Santa Cruz, California, USA), NPY (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), NPY1R (1:500; Abcam), type I collagen α1 chain (COL1A1; 1:300; CST, Boston, MA, USA), runt-related transcription factor 2 (Runx2; 1:1000; Bioss, Beijing, China) and GAPDH (1:1000; Key-GEN, Nanjing, China) overnight at 4°C. Membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies, and the proteins were visualized using enhanced chemiluminescence (Beyotime Institute of Biotechnology). The expression of target genes was evaluated using gray value analysis, which was normalized to the GAPDH reference gene.
3
Western Blotting Antibody Validation
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Primary antibodies used for Western blotting were GAPDH (Santa Cruz Biotechnology, sc-3233), RNA polymerase II (Pol II; Santa Cruz Biotechnology, sc-56767); Cleaved PARP1 (Cell Signaling Technology, 5625), p-Ser2 RNAPII (Rbp1; Cell Signaling Technology, 13499), p-Ser5 RNAPII (Rbp1; Cell Signaling Technology, 13523), CCND1 (Cell Signaling Technology, 2922), EZH2 (Cell Signaling Technology, 5246), Tri-Methyl-Histone H3 (Lys27; Cell Signaling Technology, 9733), Histone H3 (Cell Signaling Technology, 3638); EWS-FLI1 (Abcam ab15289), NPY1R (Abcam ab91262); Acetyl-Histone H3 (Lys27; 39133). Secondary antibodies used were mouse IgG (GE Healthcare Life Sciences NXA931) and rabbit IgG (GE Healthcare Life Sciences NA934) GE Healthcare. Mouse or rabbit IgG (sc-2025 or sc-2027) for chromatin immunoprecipitation was from Santa Cruz Biotechnology. GSK-J4 and THZ1 were from Abmole.
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