Pclipf

The pHaloTag–GR has been previously described [11 (link)]. Briefly, the construct expresses the rat GR with HaloTag protein (Promega, Madison, WI, USA) fused in the C-terminal domain under the CMVd1 promoter. The SNAP-tag–GR expresses the rat GR with SNAP-tag protein fused in the C-terminal domain under the CMV promoter. This was generated by PCR amplification from HaloTag–GR and sub cloned into the pSNAPf (N9183S, New England Biolabs, Ipswich, MA, USA) backbone with NheI and AgeI sites. The CLIP-tag–GR expresses the rat GR with CLIP-tag fused in the C-terminal domain under the CMV promoter. This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites. The mEOS3–GR was generated by purification of the rat GR sequence from a AgeI/XhoI digested fragment of the pEGFP–GR vector [48 (link)], and subsequent sub-cloning into a pre-digested AgeI/XhoI pmEOS3 plasmid, kindly provided by the Lippincott-Schwartz lab. Finally, pEGFP-NF1[49 (link)] was used as an homogenous nuclear marker in Figure 3.

Most plasmids were purchased from Addgene or were shared by independent labs. GFP-tagged proteins candidates were selected based on gene ontology annotation containing terms “identical protein binding” (GO:0042802), “protein homotrimerization” (GO:0070207), “protein trimerization” (GO:0070206), “protein dimerization” (GO:0046983) and “protein tetramerization” (GO:0051262) and independently verified for their self-interacting ability by the tool SLIPPER (http://lidong.ncpsb.org/slipper/index_1.html). The resulting pools of proteins were selected to cover a range of valences. The proteins tested in each class are: monomeric, p53; dimeric, AKT, Rac2; trimeric, HSF1; tetrameric, PKM2; octomeric, PAICS; IDR-containing: FUS, TDP-43. pcDH-Halo-DCP1A, pcDH-SNAP-DCP1A, pcDH-GFP-DCP1A, pcDH-mCherry-DCP1A, and pcDH-CLIP-DCP1A were constructed by first sub-cloning the DCP1A open-reading frame (ORF) from pEGFP-DCP1A into the pcDH backbone to generate pcDH-DCP1A. The ORFs of Halo, SNAP, GFP, mCherry and CLIP were PCR amplified from pFN21A (Promega), pSNAPf (NEB), pEGFP-C1 (Clontech), pEF1a-mCherry (Clontech), and pCLIPf (NEB), respectively. These amplicons were then sub-cloned into the pcDH-DCP1A backbone to generate the appropriate plasmids.