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VDR translocation assay

ZL Zongtao Lin
HC Hao Chen
AB Anna Y. Belorusova
JB John C. Bollinger
ET Edith K. Y. Tang
ZJ Zorica Janjetovic
TK Tae-Kang Kim
ZW Zhongzhi Wu
DM Duane D. Miller
AS Andrzej T. Slominski
AP Arnold E. Postlethwaite
RT Robert C. Tuckey
NR Natacha Rochel
WL Wei Li
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The effects of 1,20 S(OH)2D3 on VDR translocation from the cytoplasm to the nucleus were tested on the previously described SKMEL-188 cell model21, 33, using cells stably transduced with pLenti-CMV-VDREGFP-pgk-puro (VDR and EGFP expressed as fusion protein)34. Cells were treated with secosteroids (up to 100 nM) for 90 min followed by analysis with Cytation 5 (BioTek, Winooski, VT, US). Translocation to the nucleus was determined by counting cells with a fluorescent nucleus and the results are presented as the percentage of the total cells that displayed nuclear staining, as described previously21. The data were obtained from at least two separate experiments, with images taken in the central area from at least three different wells and counted as described21, 34.

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