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. 2018 Mar;24(3):381–395. doi: 10.1261/rna.064584.117

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© 2018 Sgromo et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 6. — The CBM is necessary for Bam-mediated mRNA repression. (A) Tethering assay using the F-Luc-5BoxB reporter and λN-HA-tagged Bam (wild-type or the indicated mutants) in S2 cells. The samples were analyzed as described in Figure 1B. (B) Northern blot of representative RNA samples shown in A. (C) Western blot showing the equivalent expression of λN-HA-tagged proteins used in A and B. (D) SBP pull-down assay in control and CAF40-null HEK293T cells expressing V5-SBP-tagged full-length Bam. V5-SBP-tagged MBP served as a negative control. Input (1.5% for the V5-SBP tagged proteins and 1% for endogenous CCR4–NOT subunits) and bound fractions (10% for the V5-SBP tagged proteins and 30% for the CCR4–NOT subunits) were analyzed by western blotting using the indicated antibodies. (KO) Knockout. (E) SBP pull-down assay in control and CAF40-null HEK293T cells expressing V5-SBP-tagged full-length Bam and HA-CCR4. Samples were analyzed as in D. (F) SBP pull-down assay in HEK293T cells expressing V5-SBP-tagged full-length Bam or the 4xMut. V5-SBP-tagged MBP served as a negative control. Input (1.5% for the V5-SBP tagged proteins and 1% for CCR4–NOT subunits) and the bound fractions (10% for the V5-SBP tagged proteins and 30% for CCR4–NOT subunits) were analyzed by western blotting using the indicated antibodies. (G) SBP pull-down assay in HEK293T cells expressing V5-SBP-tagged full-length Bam or the 4xMut and HA-tagged CCR4.