Skip to main content
Fig. 1 | Genome Biology

Fig. 1

From: Control of immediate early gene expression by CPEB4-repressor complex-mediated mRNA degradation

Fig. 1

Poly(A) RNA interactome capture upon HDAC inhibition by RMD. A Schematic illustrating the poly(A) RNA interactome capture procedure. Cells were chemically crosslinked with 0.05% formaldehyde, and after cryogenic lysis, poly(A) RNA was affinity purified using an oligo d(T)25 magnetic matrix. Following denaturing washes, bound proteins were eluted and subjected to LC-MS/MS. B HeLa cells were treated with the class I-specific HDAC inhibitor romidepsin (RMD, 20 nM) or an equal volume of solvent (DMSO) for 16 h prior to comparative poly(A) RNA interactome capture (n = 2). The scatter plot depicts log10-transformed label-free quantification (LFQ) values from the ensuing LC-MS/MS proteomics analysis; only proteins enriched >10-fold over non-crosslinked controls are depicted. Candidate proteins displaying an at least 2-fold increased (blue) or decreased (red) association with poly(A) RNA upon RMD treatment in two biological repeats are highlighted; n.d.: not detected. C Scatter plot depicting the fold-change in poly(A) RNA association in relation to the fold-change in mRNA abundance as determined by RNA-Seq. Only candidate proteins with increased (blue) or decreased (red) binding to poly(A) RNA upon RMD treatment are shown; Inf, infinite. D Validation of selected candidate proteins by western blot analysis after poly(A) RNA interactome capture. E Differential association of selected candidate proteins from D was quantified after normalizing to the amount of co-purified poly(A) RNA (Additional file 2: Fig. S1A). Data are presented as mean ± SD (n ≥ 2), indicated p-values were calculated using a two-sided, one-sample t-test; * p < 0.05, ** p < 0.01

Back to article page