Proliferation was measured using the Celltiter Glo assay (Promega, Madison, WI). Briefly, a total of 1000 cells per well were seeded in sextets on a 96 well plate, and luminescence was measured in a Modulus plate reader (Turner Biosystems, Sunnyvale, CA) at 2 and 72 hours. Fold change was calculated by comparing luminescence reading between the two time points. Cells treated with 300 nM rElafin or rSLPI (R&D systems, Minneapolis, MN) were grown in medium containing only 0.5% FBS, otherwise cells were cultured as described above. In the antibody blocking experiment, 200 ng/ml of affinity-purified Elafin antibody or IgG purified from pre-immune sera of the same rabbit was used.
Relafin
Manufactured by R&D Systems
Sourced in United States
RElafin is a recombinant human protein that belongs to the elafin family of serine protease inhibitors. It functions as an inhibitor of elastase and proteinase 3.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using relafin
1
Elafin and SLPI Proliferation Assay
2
Elafin and SLPI Proliferation Assay
3
EGFR Dimerization and Internalization Assay
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After starvation for 12 h, wild-type HCC cells were stimulated with 10 μg/ml recombinant Elafin (rElafin, R&D Systems, MN, USA) or 20 ng/ml EGF (236-EG, R&D Systems, MN, USA) or with BSA (Biofroxx, Germany) as the control for 30 min. After that, the cells were treated with the crosslinking reagent BS3 reagent (Thermo Fisher, MI, USA) at a concentration of 3 mM for 30 min at room temperature. Then, 40 μL 1 M Tris-HCl pH 7.5 was added for an additional 15 min to terminate the reaction. Finally, the cells were lysed and subjected to western blotting to assess EGFR dimerization. The dimers were represented by bands at approximately 300 KD, while EGFR monomers were represented by bands at 170 KD.
To assess the internalization of EGFR, after starvation for 12 h, the cells were stimulated with 10 μg/ml recombinant Elafin or 20 ng/ml EGF or BSA for 30 min. Then, the cells were subjected to immunofluorescence (IF) to assess the cell surface expression of EGFR.
To assess the internalization of EGFR, after starvation for 12 h, the cells were stimulated with 10 μg/ml recombinant Elafin or 20 ng/ml EGF or BSA for 30 min. Then, the cells were subjected to immunofluorescence (IF) to assess the cell surface expression of EGFR.
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