Figure 3

(a) Outline of RIP-RT and controls. hES/HA-DND1 cells were treated with dox (+DOX) or not treated with dox (-DOX). The cells were used to make mRNP lysates and the input fraction was incubated with anti-HA antibody or as control, anti-FLAG antibody for RIP. Excess tRNA was added to the input. RNA extracted from the RIP fraction was used for RT-PCR. (b) Defining the pluripotency factors which are targets of DND1. Anti-HA antibody (a-H) or anti-FLAG (a-F) was used for RIP. After RIP, RT-PCR was performed on equal amounts of cDNA from I, S and RIP fractions. RT+ indicates presence of Reverse Transcriptase during cDNA preparation. RT- are control lanes with no Reverse Transcriptase. Specific pull down of OCT4, SOX2 and NANOG by anti-HA antibody is observed in lane 13. The PCR cycle number used to detect each transcript is described in Methods. (c) Transcripts encoding pluripotency factors and cell cycle regulators targets are targets of DND1. RT-PCR of the S fraction was not performed on these samples. Specific pull down of LIN28, TP53 and LATS2 by anti-HA antibody is observed in lane 7. (d) Pro-apototic factor BAX and anti-apoptotic factor BCLX are specifically pulled down with HA-DND1 during RIP using anti-HA antibodies (lane 7).