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Figure 1 | BMC Molecular Biology

Figure 1

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

Figure 1

DND1 expression in hES cells. (a) Immunoblotting using anti-DND1 antibody of MEF, hES line H9, mouse testes (T) and spleen (S) (10 μg protein/lane). Control (C) is 293T cells transfected with HA-DND1 encoding expression vector. (bottom) Blot reprobed with anti-GAPDH. (b) RT-PCR to amplify DND1 in human testes (T), hES cells H1, H9, and MEFs. (+) lanes had superscript; (-) had no superscript during RT. (bottom) RT-PCR for HPRT. (c) . The first virus expresses rtTA that binds dox. The second virus carries dox-responsive promoter (sgTRE) that drives HA-DND1 expression. Dox also induces GFP expression, which allows identification of cells carrying both constructs. att (sites for recombinational cloning); LTR and ΔLTR (long terminal repeats); IRES (internal ribosome entry site). (d) FACS analysis indicates hES/HA-DND1 cells maintain high expression (77% +ve) of OCT4 and SSEA-4 (quadrant Q2, top right). (e) hES/HA-DND1 cells induced to express HA-DND1 upon dox treatment (100 to 1000 ng/mL) for 24 h. Immunoblotting using anti-HA antibody (top panel), anti-DND1 antibody (middle panel) and anti-GAPDH antibody (bottom panel) (20 μg hES/HA-DND1 lysates/lane). Relative HA-DND1 levels (fold HA) range from 1.6 - 3.7 fold with increasing dox. (Relative HA-DND1 of each lane is ratio of HA-DND1/GAPDH). The level of endogenous DND1 (DND1/GAPDH) in hES/HA-DND1 cells treated with 0 to 1000 ng/mL dox was 1.2, 0.9, 1.1, 1.1 and 0.7, respectively. The level of induced HA-DND1 to endogenous DND1 levels (HA/endog) was estimated to be 1.7 to 5.4-fold, with increasing dox.

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