Fig. 3

IRES-mediated generation of estrogen receptor alpha (ERα)46. a A representative image of a Western blot of extracts from MDA-MB-231 cells transfected with constructs expressing either ERα46, wild-type ERα66, or an ERα66 cDNA harboring mutated ATG174/176 codons (ERα46 ⁰), illustrating the loss of expression of ERα46 in cells transfected with the vector encoding ERα46⁰. b Scheme illustrating the bi-cistronic pTRIP-hERaAB-L2 vector harboring the Renilla luciferase (LucR) and Firefly luciferase (LucF(2CP)) cistrons separated by the sequence coding for the A/B domains of the human ERα66. c, d Stress-induced alternative initiation of translation. c MDA-MB-231 cells transduced with lentivirus expressing the LucR-hERaAB-LucF construct were subjected to 6 h of stress with 1,4-dithiothreitol (DTT; 3, 5, or 8 mM), thapsigargin (Tg; 10, 50, or 500nM) or vehicle control (ctrl). Reporter gene activities from three independent experiments in duplicates are shown as the LucF/LucR ratio. d Representative Western blot of the increased expression of ERα46 observed in stressed MDA-MB-231 cells transfected with cDNA expressing ERα66. *P < 0.05, **P < 0.01, ***P < 0.001, versus control (Kruskal-Wallis) ns not significant