Technical Reference #1929

1929.

Modulation of ERa transcriptional activity by the orphan nuclear receptor ERRb and evidence for differential effects of longand shortform splice variants Vincent Bombail; Frances Collins; Pamela Brown; Philippa T.K. Saunders, The Queen's Medical Research Institute, Molecular and Cellular Endocrinology, 314(1929), (2010)
Link To Paper

Keywords:
Oestrogen receptor; Orphan receptor; F-domain; Splice variant; ESRRB

Materials & Methods:
Ishikawa cells (4×105) were plated in glass bottom dishes (MatTek Ashland MA USA). Cells were transfected with 2mg of plasmid DNA using Jet PEI (as above); plasmids expressed ERRbS or ERRbL tagged at the Nterminus with yellow fluorescent protein (YFPERR bS YFPERR bL). Transfection efficiency was routinely ∼30%. The intranuclear mobility of YFPERR bS and YFPERR bL was examined using Fluorescence recovery after photobleaching (FRAP) on an LSM510 metaconfocal (Zeiss) with a stage maintained at 37 ◦C. Prior to imaging the media was replaced the PBS with 10mM HEPES buffer (Sigma). In each cell two areas within the nucleus were selected and fluorescence was measured before and after bleaching of one of the two areas. Recovery of fluorescence was monitored every 3 s for 30 s. The fluorescence intensity data was normalised for each cell and used in a nonlinear regression model Y = Ymax ×(1−e−Kx) (GraphPad Prism 4) where the regression coefficient r2 was typically 0.95. The Ymax and halflife of recovery values (0.69/K) were averaged for at least 20 cells per treatment.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
Ishikawa cells