Fig. 4

Klf4 glutamylation impedes K48-linked ubiquitination to sustain its stability. a Ccp1- and Ccp6-deficient MEFs were treated with cycloheximide (CHX) (20 μg/ml) for the indicated time and followed by immunoblotting. Percentage of residual protein levels was counted as means ± S.D. b Ccp6^+/+ or Ccp6^−/− MEFs were transfected by OSKM for 2 days and treated with MG132 (10 μM) for 4 h. Cells were collected and immunoprecipitated with anti-Klf4 antibody followed by detection with anti-poly-Ub antibody. c Klf4 ubiquitination is K48-linked but not K63-linked. pMX5-Klf4 together with pCDNA4-MycHis-Ubiquitin (Ub) or Ub(K48R), Ub(K63R) mutant were transfected into 293T cells for 48 h and analyzed as in b. d rGST-Klf4 was pre-incubated with lysates from 293 T cells transfected with Flag-TTLL4 at 37 °C for 2 h, followed by GST pulldown with Flag-βTrCP1 and immunoblotting with indicated antibodies. e rGST-Klf4 was pre-incubated with lysates from 293T cells transfected with Flag-TTLL4 at 37 °C for 2 h. Klf4 protein was then incubated with recombinant active ERK1 (Millipore) and 2 mM ATP at 30 °C for 30 min. His-tagged E1 (Uba1), E2 (UbcH5c), ubiquitin, Rbx, Flag-tagged βTrCP1, Skp1, Cullin1 and GST-tagged Klf4 were incubated for the in vitro ubiquitination reconstitution assay at 30 °C for 2 h. Ubiquitinated Klf4 was probed by anti-GST antibody. f TTLL4 deficiency promotes the ubiquitination of Klf4 in OSKM-induced MEFs. Ttll4^+/+ or Ttll4^−/− MEFs was transfected by OSKM for 2 days and assessed for ubiquitination as described in b. g Ttll4-deficient MEFs were treated with cycloheximide (CHX) (20 μg/ml) for the indicated time, followed by immunoblotting as described in a. h Ttll4-deficient 4- to 8-cell stage embryos were isolated and immunostained with anti-Klf4 and GT335 antibodies. Scale bar, 20 μm.For Ttll4^+/+ embryos, n = 67. For Ttll4^−/− embryos, n = 55. All data are representative of four independent experiments