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. Author manuscript; available in PMC: 2014 Feb 28.
Published in final edited form as: Cell. 2013 Feb 28;152(5):1037–1050. doi: 10.1016/j.cell.2013.02.006

Figure 6. Jmjd3 Targets PHF20 for Ubiquitination by Recruiting an E3 Ligase Trim26.

Figure 6

(A) 293T cells transfected with HA-tagged ubiquitin, Flag-tagged PHF20 and HA-tagged Jmjd3. Cell lysates were immunoprecipitated with anti-Flag beads, followed by IB with an anti-ubiquitin antibody. (B) 293T cells transfected with Flag-tagged PHF20, HA-tagged Jmjd3 and different Trim26 shRNA constructs as indicated. Cell lysates were immunoprecipitated with anti-Flag beads and analyzed with an anti-K48 ubiquitin. (C) Number of AP-positive colonies counted on day 14 of 4F-mediated reprogramming of WT and PHF20-deficient MEFs transduced with Trim26-specific or control shRNAs. Asterisks indicate significant differences between groups (*p < 0.05, **p < 0.01, N.S. p > 0.05). (D) Western blot analysis of Jmjd3, Trim26 and PHF20 expression in WT and Jmjd3-deficient MEFs during cellular reprogramming. (E) 293T cells transfected with HA-Trim26, Flag-PHF20 and HA-Jmjd3 as indicated. Cell lysates were immunoprecipitated with anti-Flag beads, followed by IB with an anti-K48 ubiquitin antibody. (F) 293T cells transfected with Flag-Trim26, GFP-PHF20 and HA-Jmjd3 as indicated. Cell lysates were immunoprecipitated with anti-Flag beads, followed by IB with PHF20 and HA antibodies. (G) 293T cells transfected with Myc-Trim26 and HA-Jmjd3 constructs. Cell lysates were immunoprecipitated with anti-HA beads, followed by IB with an anti-Myc antibody. (H) 293T cells transfected with Flag-PHF20 and HA-Jmjd3 constructs. Cell lysates were immunoprecipitated with anti-Flag beads, followed by IB with an anti-K48 ubiquitin antibody. See also Figure S6.