DEVELOPMENTAL BIOLOGY. For the article “Requirement of Nanog dimerization for stem cell self-renewal and pluripotency,” by Jianlong Wang, Dana N. Levasseur, and Stuart H. Orkin, which appeared in issue 17, April 29, 2008, of Proc Natl Acad Sci USA (105:6326–6331; first published April 24, 2008; 10.1073/pnas.0802288105), the authors note that, as indicated by thin vertical lines, multiple blots from the same experiments were juxtaposed in Fig. 1A, and irrelevant lanes were removed from the same blot in Figs. 1B, 3
Fig. 1.
Confirmation of Nanog–Nanog interaction (dimerization). (A) Western blot analysis of gel filtration fractions containing protein complexes using anti-Nanog antibody. Fractions that may contain Nanog homodimers are indicated with a red rectangle (Upper). A close-up of fractions containing potential Nanog dimers and monomers is also presented (Lower). Sample fractions were loaded in four (Upper) and two (Lower) denaturing polyacrylamide gels, respectively, and Western data are presented with thin vertical lines separating the different gels. (B) CoIP in heterologous 293T cells. 293T cells were transiently transfected with constructs indicated, and total lysates were prepared and incubated with anti-M2-FL agarose (Left) or anti-V5 agarose (Right) overnight. Unbound material was washed away; bound material was eluted and subjected to Western blot analyses using the antibodies indicated (Upper). Total lysates as input were also subjected to Western blot analyses with anti-V5 antibody (Lower). < n.s. indicates a nonspecific signal; * indicates FL-Nanog. The thin vertical lines indicate removal of irrelevant lanes and juxtaposition of the two lanes relevant for this study.
Fig. 3.
Nanog homodimerization is required for its interaction with other pluripotency factors. (A) CoIP of V5his-tagged pluripotency network proteins Sall4, Zfp281, Zfp198, and BirA (control) with dimeric FLbioNanog and monomeric FLbioNanog10WA mutants. (B) CoIP of V5his-tagged Dax1 and BirA (control) with dimeric FLbioNanog and monomeric FLbioNanog10WA mutants. < n.s. indicates a nonspecific signal; * indicates the FLbioNanog signal. Note the much lower input level of FLbioNanog than FLbioNanog10WA and yet a higher IP signal (*) of FLbioNanog than FLbioNanog10WA. (C) CoIP of V5his-tagged Nac1 with dimeric FLbioNanog and monomeric FLbioNanog10WA mutants. (D) CoIP of V5his-tagged Oct4 with dimeric FLbioNanog and monomeric FLbioNanog10WA mutants. The thin vertical lines indicate removal of irrelevant lanes and juxtaposition of the two lanes relevant for this study.D, and 4B. In accordance with PNAS editorial policy on the preparation of digital images, the corrected figures and legends appear below. These changes do not affect the conclusions of the article.
Fig. 4.
Tethered Nanog mutants maintain DNA-binding capacity. (A) Schematic depiction of the tethered Nanog dimer (NN) and monomer (NNH) mutants. The 22-aa flexible polypeptide linker is shown. (B) EMSA of mutants' binding to the Cdx2 (lanes 1–9) and the Gata6 (lanes 10–13) promoter DNA sequences. * indicates specific binding signals; < denotes the supershift bands by anti-Nanog. Wild-type (wt) and vector-transfected COS extracts were used as negative controls. The thin vertical line indicates removal of irrelevant lanes and juxtaposition of lanes relevant for this study.