Fig 1: CID proteins colocalize with pSer2 and pThr4 Pol II.(A) Representative confocal fluorescent images of HA-RPRD1B full length or HA-RPRD1B ΔCID (red), pSer2 or pThr4 Pol II (green), and DAPI (blue) in HEK293 cells. Scale bars, 5 μm. Profile intensity plots between red and green channels are shown. (B) Confocal fluorescent images of HA-CHERP full length (red) and pSer2/pThr4 Pol II (green). Scale bars, 5 μm. (C) Confocal fluorescent images of HA-RPRD1B (red) and 52X T4A Pol II or 52X WT Pol II (yellow). Scale bars, 5 μm. Profile intensity plots between red and green or red and yellow channels are shown. All IF experiments were performed three independent times. (D) Anti-YFP coimmunoprecipitation of T4A Pol II or WT Pol II with HA-RPRD1B. Representative blots are shown, and quantification is based on three independent biological replicates. Plot shows means with SD. IB, immunoblot; IP, immunoprecipitation; WCL, whole-cell lysate. (E) Distribution of the input normalized ChIP signal of HA-RPRD1B or HA-CHERP across human annotated genes (27). Average peak signal between a window 2 kb upstream/downstream from the TSS/TES. *P < 0.05.
Fig 2: CID proteins recognize both pSer2 and pThr4 CTD.(A) FA measurements of the CID of RPRD1B with FITC- labeled pS2/pT4 CTD peptides. (B) 2Fo-Fc electron density map (contour to 1.0 s) of RPRD1B’s CID complexed with the pThr4 CTD peptide. (C) Side-by-side view of RPRD1B binding to the pThr4 or pSer2 CTD peptide (PDB: 4Q94). Conserved Arg106 and Arg114 are shown with stick representation. (D) FA measurements of WT or mutant CID of SCAF4, RPRD1A, and RPRD2 with pS2/pT4 CTD peptides. Experimental isotherms were fitted to a total binding model. Binding assays were performed in triplicate. Error bars indicate the SD. (E) Structural modeling of the conserved recognition of CID of SCAF4 (pink), RPRD1A (blue), and RPRD2 (light blue) binding to the pThr4 CTD peptide.
from Cell Signaling Technology for RPRD1B Antibody