Figure 1
From: Baf53a is involved in survival of mouse ES cells, which can be compensated by Baf53b
Baf53a is expressed in ES cells, and Baf53a interacts with the POU-specific domain of Oct3/4. (A) Expression of Baf53a mRNA in E14 ES cells cultured with or without LIF for 3–6 days. Expression of Baf53a and markers of undifferentiated cells (Dax1, Rex1, and Zfp57) mRNA was examined by quantitative RT-PCR. (B) Expression of Baf53a mRNA after downregulation of Oct3/4. ZHBTc4 ES cells were cultured with or without 1 μg/mL tetracycline (Tet) for 3–6 days. Expression of Baf53a mRNA was examined by quantitative RT-PCR. Dax1 was used for a positive control as an Oct3/4 target gene. These data were normalized to GAPDH, and the results are expressed as the average of three independent experiments ± the standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 (t-test). (C) Interaction between Baf53a and Oct3/4. The Myc-Baf53a expression vector was co-transfected with Flag-MBP-Oct3/4 or its truncated mutants, including C-terminal region, N-terminal region, POU domain, POU-homeo domain, and POU-specific domain, into HEK293 cells. Flag-MBP-fused proteins were pulled down using amylose resin, and the precipitates were examined by Western blot with anti-Myc antibody. Expression of each protein was confirmed with anti-Myc and anti-Flag antibodies. Arrows indicate Flag-MBP-Oct3/4 and its truncated mutants. The result shown is representative of three independent experiments. Note: ev = control Flag-MBP empty vector; Full = full length of Oct3/4 (1–352 aa); C = C-terminal region (283–352 aa); N = N-terminal region (1–130 aa); POU = POU domain (131–282 aa); Homeo = POU-homeo domain (217–282 aa); Specific = POU-specific domain (131–213 aa).
