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In an attempt to understand the possible interaction modes of the top hits in the assays above, FRED docking was performed (see Methods).
As an additional means of examining the biological potential of the APE1 inhibitors displaying the most promise in the assays above, we evaluated the ability of 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin to enhance cellular sensitivity to the alkylating agent MMS.
Following 3 days of culture in drug, plates were trypsinized and replated at equal densities into 12-well plates in complete medium, as described for cell proliferation assays above.
Aside from the targets identified in the cell-free assays above, cell-based assays suggest inhibition of the TIM22 import pathway, the GLP-1 receptor, HSP90, and tyrosyl-DNA phosphodiesterase 1 (see AIDs 493003, 540268, 540270, 624417, 686978 and 686979).
To obtain biological evidence, the effect of EAFS which has been regarded as the most effective extract among five FS extracts in the antioxidant assays above, was further estimated using the MTT assay.
Confirming the spectrofluorimetric liposome binding assays above, SPR analysis showed improved binding of SBD-TMR to the same liposomes immobilized on a Dextran-coated gold chip, as the concentration of GD1a was increased.
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We assayed expression levels in a subset of 9 organs and two stress conditions that were assayed above.
The assays described above were conducted in 20 mM Tris buffer (pH 8.0,10%% glycerol).
Cell aggregation assays confirmed the above results.
In 61% of the above assays, crickets left the tube.
These quantifying results are consistent with the PCR assays mentioned above.
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