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Figure 2 | BMC Molecular Biology

Figure 2

From: Transcripts that associate with the RNA binding protein, DEAD-END (DND1), in embryonic stem (ES) cells

Figure 2

The RIP-RT procedure. (a) Outline of RIP-RT procedure. hES/HA-DND1 cells were treated with and without dox. Anti-HA antibody was used to pull-down HA-DND1 together with associated mRNA. The mRNA was eluted and RT-PCR used to amplify and detect P27. (b) Details of the RIP procedure as described in [29]. hES/HA-DND1 cells were used to prepare lysates (mRNP lysates). A fraction of the lysate was used as input (I) for immunoprecipitation (IP) using anti-HA antibody linked to beads. After an overnight incubation, the input was centrifuged. The supernatant (S) was removed. RNA was extracted from the beads (RIP fraction), I and S fractions and used for RT-PCR. (c) PCR for P27 and HPRT on the input (I), supernatant (S) and immunoprecipitation (RIP) fractions. ES cells treated with (+) and without (-) dox were used. Each fraction of the ES cells with and without dox treatment was used to generate cDNA. The RT: + represents presence of Reverse Transcriptase and the RT: - represents no Reverse Transcriptase (control) during cDNA synthesis. (d) P27 levels in input fraction of untreated (dox-) and treated (dox+) cells when higher PCR cycle numbers (35 cycles) are used. (e) (top) Immunoblotting for HA-DND1 in the mRNP lysates, input and supernatant fraction of ES cells treated with (+) or without (-) dox. Control (C) was 293T cells transfected with HA-DND1 encoding expression vector. (bottom) Blots were reprobed with GAPDH. Relative HA-DND1 levels (HA-DND1/GAPDH ratios) in the fractions are indicated at the bottom in blue.

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