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. 2013 Mar 21;9(3):e1003397. doi: 10.1371/journal.pgen.1003397

Figure 4. NM1 C-terminus and intact motor function are required for the association of NM1, SNF2h, and actin with rRNA genes.

Figure 4

(A) ChIP assays performed on HEK293T cells or HEK293T cells constitutively expressing V5-tagged wt and mutated NM1 constructs (as indicated) were analyzed by qPCR. The bars diagrams show the relative amounts of rDNA promoter, 18S rDNA and 28S rDNA precipitated with V5 antibodies. The values are presented as the percentage of the input signal for each pair. Error bars represent standard deviations. (B) Steady state expression levels of endogenous WSTF and SNF2h analyzed on immunoblots of total cell lysates from HEK293T cells expressing V5-wtNM1, V5-RK605AA NM1 and V5-ΔC NM1 mutants. WSTF and SNF2h steady state expression levels were normalized against endogenous actin levels. Expressions of V5-wtNM1, V5-RK605AA NM1 and V5-ΔC NM1 mutants were monitored with anti-V5 antibodies. (C–E) ChIP assays on chromatin from HEK293T cells stably expressing V5-wtNM1, V5-RK605AA and V5-ΔC NM1 mutants with antibodies against V5, WSTF, SNF2h and actin. qPCR analysis was performed with primers amplifying promoter and 18S rDNA. The values are presented as the percentage of the input signal for each pair. Error bars represent standard deviations. (F–G) ChIP assays on chromatin from HeLa cells untreated (F) or treated with BDM (G) using the indicated antibodies (see below the bars). qPCR analysis was performed with primers amplifying rRNA gene promoter, 18S and 28S rDNA as well as IGS. The values are presented as the percentage of the input signal for each primer pair. Error bars represent standard deviations.