Mononuclear cells from the controls and primary AML patients were washed twice with phosphate buffer saline and resuspended in 200 μl of lysis buffer containing a mix of protease inhibitors (Beyotime, Haimen, Jiangsu, China). Immunoblots were prepared as previously described [22 (link)]. Rabbit anti-GDF15 monoclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-β-tubulin antibody (Abcam, Cambridge, MA, USA), rabbit anti-Cdk2 antibody (Abcam, Cambridge, MA, USA), rabbit anti-Cyclin D1 antibody (Abcam, Cambridge, MA, USA), rabbit anti-P21 antibody (Abcam, Cambridge, MA, USA) and rabbit anti-β-actin monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA). The THP-1 cell CM was obtained from cells cultured in regular medium with 1% FBS at different cell densities (2 × 105/ml, 5 × 105/ml, 1 × 106/ml, 2 × 106/ml). The ELISA analysis for GDF15 was performed according to the manufacturer’s instructions (R&D systems).
Rabbit anti cyclin d1 antibody
Manufactured by Abcam
Sourced in United States
Rabbit anti-Cyclin D1 antibody is a primary antibody that specifically binds to the Cyclin D1 protein. Cyclin D1 is a regulatory protein involved in the cell cycle and its expression is often associated with cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Rabbit anti p21 antibody, by Abcam (1 mentions)
Rabbit anti β actin monoclonal antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti β tubulin antibody, by Abcam (1 mentions)
Rabbit anti gdf15 monoclonal antibody, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Beyotime (1 mentions)
Rabbit anti c myc antibody, by Bioss Antibodies (1 mentions)
Goat anti rabbit secondary antibody, by Bioss Antibodies (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
3 protocols using rabbit anti cyclin d1 antibody
1
Immunoblot Analysis of AML Mononuclear Cells
2
Protein Expression Analysis via Western Blot
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RIPA lysis buffer (Beyotime, Shanghai, China) was used for protein extraction, and the BCA Protein Assay Kit was used to measure the amount of protein (Solarbio, Beijing, China). Total proteins were then subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electrotransferred to PVDF membranes (Bio-Rad, Hercules, CA, United States). Nonspecific binding sites were blocked using 5% nonfat milk for 2 h and the membranes were incubated at 4 °C overnight with rabbit anti-Wnt2 antibody (1:1000; Affinity, Melbourne, United States), rabbit anti-c-myc antibody (1:1000; Bioss, Beijing, China), rabbit anti-CD44 antibody (1:1000; Affinity, Melbourne, United States), rabbit anti-cyclin D1 antibody (1:1000; Abcam, Cambridge, United Kingdom), and rabbit anti-β-tubulin antibody (1:4000; Proteintech, Chicago, United States). The PVDF membranes were then treated for 1 hour with a goat anti-rabbit secondary antibody that was HRP conjugated (1:5000; Bioss, Beijing, China). Enhanced chemiluminescence reagents (Beyotime, Shanghai, China) were used to visualize the bands. Image J was utilized to quantify the chemiluminescent signals of protein bands using β-tubulin as an internal control.
3
Western Blot Analysis of Developmental Proteins
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The cells or embryo brain tissues were lysed with RIPA lysis buffer (Solarbio, Beijing, China) supplemented with a protease inhibitor cocktail (Sigma) and 10 mM PMSF (Solarbio). Following centrifugation at 12,000 g for 20 min at 4 • C, the concentration of protein was measured using BCA Protein Assay kit (Thermo Scientific). Subsequently, equal amount of protein was subject to 12% SDS-PAGE and transferred to nitrocellulose membranes using semi-dry electrophoretic transfer method (Bio-Rad, Hercules, USA). After being blocked with 5% skim milk in PBST (PBS with 0.05% Tween-20) for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4 • C, followed by incubation with the corresponding secondary antibody at room temperature for 2 h. β-Tubulin and GAPDH were used as control. The primary antibodies used for western blot analysis were rabbit anti-Nkx2.1 antibody (1:1000; Abcam, Cambridge, USA), rabbit anti-Cyclin D1 antibody (1:1000; Abcam), mouse anti-Shh antibody (1:1000; Abcam), mouse anti-Smo antibody (1:500; Santa Cruz Biotechnology), rabbit anti-Ptch1 antibody (1:1000; Cell Signaling Technology, Boston, USA)
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