Project description:Specific protein binders with high affinity, such as antibodies or nanobodies, are scarce for most solute carrier (SLCs) transporters. Moreover, general approaches that can systematically generate binders for multi-transmembrane proteins, such as SLCs, channels or GPCRs, are still missing. Purifying and reconstituting transmembrane proteins in their lipidic environments remain challenging, hence membrane proteins normally act as poor input material for binder generation. To overcome this challenge, we first identified state-of-the-art methods to generate protein binders targeting membrane transporters. Next, we produced full length protein or cell lines as input material for 27 SLCs, which were then processed in the selected binder generation platforms. As a result, we obtained >300 binders for 23 SLCs, 1 SLC failed and 3 SLCs are still in process. Then we established a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. To further highlight the suitability of high-performance binders as an important tool for the biomolecular characterization of SLCs, we tested whether three SLC12A6 binders could be used in a quantitative co-immunoprecipitation followed by mass spectrometry (IP-MS) approach. This initial analysis of the IP-MS dataset showed that SLC12A6 specific binders are capable of strongly enriching SLC12A6. We also showed that despite high sequence similarity, the different binders showed different binder to target ratios, indicating different affinities to SLC12A6 in cell lysates. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family.
Project description:Characterization of AGO2 bound short RNAs from nuclear extracts of HeLaS3 cell line. Nuclei were isolated from HeLaS3 cells; a fraction of Nuclear lysates was harvsted and sequenced as the 'input' samples. The rest of nuclear lysates were Immunoprecipitated using AGO2 monoclonal antibodies or Isotype matched IgG. Following immunoprecipitation, RNA was extracted from AGO2 or control IgG IP for short RNA sequencing. Two independent biological replicates were carried out.
Project description:Characterization of AGO1 bound short RNAs from nuclear extracts of HeLaS3 cell line. Nuclei were isolated from HeLaS3 cells; a fraction of Nuclear lysates was harvsted and sequenced as the input samples. The rest of nuclear lysates were immunoprecipitated using AGO1 monoclonal antibodies or Isotype matched IgG. Following immunoprecipitation, RNA was extracted from AGO1 or control IgG IP for short RNA sequencing. Two independent biological replicates were carried out.
Project description:Characterization of AGO2 bound short RNAs from nuclear extracts of Jurkat cell line. Nuclei were isolated from cells; a fraction of Nuclear lysates was harvested and sequenced as the input samples. The rest of nuclear lysates were Immunoprecipitated using AGO2 monoclonal antibodies or Isotype matched IgG. Following immunoprecipitation, RNA was extracted from AGO2 or control IgG IP for short RNA sequencing. Two independent biological replicates were carried out.
Project description:Immunoprecipitation-mass spectrometry (IP-MS) was performed in Calu-6 (SMARCA4-WT) and NCI-H1693 (SMARCA4-del) cell lines treated with the SMARCA2-selective degrader PRT3789 for 6 or 48 hours. SWI/SNF complexes were immunoprecipitated using an antibody against SMARCC1 and analyzed by LC-MS/MS.
Project description:Primordial germ cells (PGCs) are the embryonic precursors to egg and sperm. When removed from the embryonic gonad, PGCs can give rise to embryonic germ cell lines (EGs), pluripotent stem cells that display most of the characteristics of embryonic stem cells (ESCs) including the ability to form teratomas and to contribute to chimera formation. In mice, EG cells can be derived between E8.5 up to E12.5 of embryonic development, at which point the PGCs undergo sexual differentiation and in the male transition into unipotent gonocytes. Dazl, a germ cell-specific RNA-binding protein, is specifically expressed in developing PGCs and is required for proper germ cell development. Dazl knockout mice are infertile, but the molecular mechanisms underlying this phenotype are still unknown. Here we demonstrate that Dazl localizes in granular structures in mammalian PGCs but not in ESCs. We demonstrate Dazl plays a central role in a large mRNA/protein interactive network that includes members of Fragile-X family RNA-binding proteins. We demonstrate that Dazl and Fxr1 play a central role in these granules and directly regulate the translation of specific core pluripotency factors, including Sox2 and Suz12. RNA species interacting specifically with Dazl in primordial germ cells were identified by RNA-IP microarray analysis. This dataset contains data from UV-crosslinked RNA-IPs from in vitro PGC lysates. UV-crosslinked RNA-IP (anti-GFP and anti-PABP1) experiments from in vitro derived primordial germ cells expressing Dazl-GFP-V5. Input Total RNA and polyA-binding protein 1 (PABP1) IP were used as controls. Control and IP-enriched RNA samples were analyzed by Agilent Mouse Whole Genome 4X44K one-color microarrays. Two replicates for each condition were done.
Project description:For the detection of tiRNAs accumulated in Cyt c-IP vs tiRNAs in total cell lysates during hyperosmotic stress in the presence of angiogenin Total cell lysates from mouse embryonic fibroblasts treated with angiogenin under hyperosmotic stress. Lysates split into two parts: one part serveed as the total RNA, the other part served as Cyt c-IP fraction. Small RNAs were enriched from both parts and went through deep sequencing followed by bioinformatic analysis.