Oct-4RR has equivalent transactivation potential in undifferentiated and differentiated cellular environments

Copyright information:Taken from "Use of altered-specificity binding Oct-4 suggests an absence of pluripotent cell-specific cofactor usage"Nucleic Acids Research 2005;33(18):6011-6023.Published online 20 Oct 2005PMCID:PMC1266064.© The Author 2005. Published by Oxford University Press. All rights reserved Undifferentiated P19 (P19U) cells or P19 cells induced to differentiate with 0.5 µM RA for 4 days (dP19 cells) were transfected with specified plasmids. ( and ) Reporter plasmid was transfected at 120 and 60 ng for P19U and dP19, respectively. Extract protein concentration was measured and half as much total protein was used in experiments for dP19 than P19U. Fold activation refers to the proportional increase in reporter gene expression in response to Oct-4RR, relative to the basal transcriptional activity of reporter vector alone. Averages and standard errors are shown for experiments repeated three times. Internal control β-gal readings, like CAT activity, seen at the higher end of Oct-4RR expression vector concentrations were seen to be depressed, probably due to squelching of transcription associated molecules or as a consequence of large amounts of mRNA overwhelming the translation process. As such, β-gal data were not used to normalize CAT experiments, but rather plotted as a separate set of representative readings for reference. Asterisk indicates over 40-fold activation of reporter gene. () For experiments using wild-type Oct-4, β-gal readings are used to standardize transcription levels. ( and ) For two of the three experiment repetitions, 20% of transfected cells were used to make whole cell extracts for EMSA and western blot experiments in (A and B). (A) An aliquot of 100 µg of protein extract was included in western blot using the anti-myc antibody against the epitope translated at the N-terminus of Oct-4RR. The predicted Oct-4 band of 38–40 kDa is shown. A blot control protein for loading and transfer normalization is shown and represents endogenous protein cross-reacting with antibody used. (B) Each EMSA reaction included 20 µg of protein extract, specified probe binding site and 1 µg of poly(dI–dC) as non-specific competitor. () Following a separate set of transfection experiments using specified plasmids, western blots were performed using antiOct-4 antibody and EMSA experiments undertaken to examine wild-type Oct-4 binding to consensus octamer site in the H2B element. At 1 µg of expression vector, bound and free Oct-4 protein extract leve