Fig. 3
From: Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice
Klf4 undergoes polyglutamylation in cell reprogramming and early embryogenesis. a MEFs were transduced with STEMCCA lentivirus for the indicated days and immunostained with anti-tubulin and GT335 antibodies. Nuclei were counterstained by DAPI. Scale bar, 10 μm. Percentages of cells with GT335 signal localized in nuclei were counted as means ± S.D. **P < 0.01. More than 100 typical cells were observed and calculated. n = 134 for D0, n = 122 for D1, n = 151 for D2. b MEFs lysates induced by OSKM for the indicated days were analyzed by immunoblotting with GT335 antibody. c Recombinant CCP6-wt and inactive CCP6 mutant (CCP6-mut) were immobilized to Affi-gel10 resin. Lysates of OSKM-induced MEFs were added for affinity chromatography. The eluted fractions were visualized by SDS-PAGE followed by silver staining. The differential band around 60 kD appeared in CCP6-mut gel was cut for mass spectrometry and identified as Klf4. d MEFs Lysates induced by OSKM for the indicated days were immunoprecipitated with anti-Klf4 antibody and analyzed by immunoblotting with GT335. e Schematic representation of four putative glutamate-rich glutamate-rich regions in Klf4 (left panel). GST-Klf4wt and indicated mutant proteins were incubated with Flag-TTLL4. GST-Klf4 proteins were pulled down by Glutathione Sepharose 4B beads, followed by immunoblotting. f CCP1- and CCP6-deficient MEFs were transduced with OSKM and stained with anti-Klf4 antibody. Nuclei were stained by DAPI. Scale bar, 10 μm. Percentages of cells with Klf4/GT335 colocalization in nuclei were counted as means ± S.D. **P < 0.01. 102 Ccp1+/+ MEF cells, 112 Ccp1−/− MEF cells, 104 Ccp6+/+ MEF cells and 117 Ccp6−/− MEF cells were observed. n = 4. g CCP6-deficient embryos were isolated and immunostained with anti-Klf4 and GT335 antibodies. Percentages of cells with Klf4/GT335 colocalization in nuclei were counted as means ± S.D. **P < 0.01.110 Ccp6+/+ embryos and 116 Ccp6−/− embryos were observed. Scale bar, 20 μm. Student’s t test was used as statistical analysis. The data represent four independent experiments. MW molecular weight marker
