Project description:The goal of this study is to compare the mRNA interactome of different RBPs in regenerating axons utilizing RNA-immunoprecipitation (RIP). Interacting mRNAs to each target RBPs were co-immunoprecipitated from axoplasm of sciatic nerve, injured 7 days ago. Interactome of each RBPs were identified by Next-generation sequencing (NGS).
Project description:In an attempt to identify proteins interacting with transcription factor E2F2, proteins extracted from HA-E2F2 overexpressing HEK293T cells were were subjected to immunoprecipitation (IP) with an anti-HA antibody or, as a negative control (NegCt), with an anti-T antibody. Immunoprecipitated proteins were eluted, digested with trypsin, and the resulting peptides were analyzed by LC-MS/MS. The whole procedure was repeated several times in an attempt to gauge reproducibility (nine replicates of the anti-HA IP and six replicates of the control IP).
Project description:Histone variant H2A.Z is a critical player in setting up the chromatin environment that mediates transcription and other activities on chromatin. However, how H2A.Z is incorporated to specific chromatin regions is not clear. To examine the potential role of sequence-specific transcription factors in targeting H2A.Z, we screened for genome-wide H2A.Z-interacting proteins in vivo using a novel technique called bait Protein-Protein Interaction-sequencing (bPPI-seq). Among the hundreds of H2A.Z-interacting proteins identified by bPPI-seq, we show that a zinc-finger transcription factor, Osr1 interacts with H2A.Z both in vitro and in vivo and co-localizes with H2A.Z on chromatin. Knockdown of Osr1 compromised H2A.Z deposition to hundreds of chromatin sites enriched with Osr1 binding motifs. Furthermore, Osr1 and H2A.Z co-regulate the expression of numerous target genes. These results indicate that Osr1 directly interacts with H2A.Z, mediates its incorporation to a large number of target sites and regulates gene expression. Our data indicate that bPPI-seq can be widely applied to identify unbiasedly interacting proteins under physiologic conditions.
Project description:293T cells were transfected with pCMV CEBPbeta LAP2 isoform. Two days post transfection, nuclear extracts were subjected to Co immunoprecipitation with mouse anti-CEBPbeta (clone H-7) or mouse control IgG using Protein G Dynabeads. After washing with RIPA buffer, beads were subjected to Mass spectrometry based protein identification to find interacting partners of CEBPbeta.
Project description:We identified the common protein interactome of rhabdomyosarcoma interacting proteins. Here we selected a subset of interactors, targeted them with sgRNA, and performed RNAseq is RH4-flag cells.
Project description:In an attempt to identify proteins interacting with transcription factor E2F2, proteins extracted from HA-E2F2 overexpressing HEK293T cells were were subjected to immunoprecipitation (IP) with an anti-HA antibody or, as a negative control (NegCt), with an anti-T antibody. Immunoprecipitated proteins were eluted, digested with trypsin, and the resulting peptides were analyzed by LC-MS/MS. The whole procedure was repeated several times in an attempt to gauge reproducibility (nine replicates of the anti-HA IP and six replicates of the control IP).