Mango Extract Effects on Cellular Pathways

After a 24 h mango extract treatment, cells were lyzed in RIPA buffer, a 1% protease and proteinase inhibitor cocktail were added (Pierce/Thermo Scientific, Rockford, IL) and centrifuged [36 (link)]. Mucosal scrapings were homogenized in a buffer (500 mM Tris–HCL, 1M Sucrose, 200 mM EDTA, 100 mM EGTA, 0.4M NaF, 10% Triton X–100, 10 mM Sodium Orthovanadate, and Protease Inhibitor Cocktail) then centrifuged at 15,000g for 10 min at 4°C, and the supernatant was stored at −80°C. Fifty microgram of protein was loaded onto the gel, followed by transfer onto PVDF membrane. Membranes were incubated with primary antibodies against NF-κB, p-NF-κB, COX-2, cleaved caspase-3, PARP-1 (Cell Signaling Technology, Beverly, MA), iNOS (Cayman Chemical, Ann Arbor, MI), PI3K (p85β), HIF-1α (Abcam, Cambridge, MA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

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Kim H., Banerjee N., Barnes R.C., Pfent C.M., Talcott S.T., Dashwood R.H, & Mertens-Talcott S.U. (2016). Mango Polyphenolics Reduce Inflammation in Intestinal Colitis—Involvement of the miR-126/PI3K/AKT/mTOR Axis In Vitro and In Vivo. Molecular carcinogenesis, 56(1), 197-207.

Publication 2016

NF-κB p-NF-κB COX-2 cleaved caspase-3 PARP-1 β-actin

Actin Antibodies Buffer Caspase 3 Cayman Cells Cox 2 Edta Egta Inos Mango Membranes Mucosal Nf κb Orthovanadate Parp 1 Pi3k Protease Protease inhibitor Protein Pvdf Ripa Sodium Sucrose Tris Triton x 100

Corresponding Organization : Texas A&M University

Other organizations : The University of Texas MD Anderson Cancer Center, Texas A&M Health Science Center

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Variable analysis

independent variables

24 h mango extract treatment

dependent variables

NF-κB
P-NF-κB
COX-2
Cleaved caspase-3
PARP-1
PI3K (p85β)
HIF-1α

control variables

RIPA buffer
1% protease and proteinase inhibitor cocktail
500 mM Tris–HCL, 1M Sucrose, 200 mM EDTA, 100 mM EGTA, 0.4M NaF, 10% Triton X–10, 10 mM Sodium Orthovanadate, and Protease Inhibitor Cocktail
Centrifugation at 15,000g for 10 min at 4°C
50 microgram of protein loaded onto the gel
Transfer onto PVDF membrane
Incubation with primary antibodies

controls

β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) as a loading control

Annotations

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