Dataset Information

GFP-Trap-IP-phospho

ABSTRACT: To analyze phosphorylation sites of ALLO-1, worm eggs expressing GFP-ALLO-1 in the atg-11 or ikke-1 background were lysed in co-IP buffer, and GFP-ALLO-1 was immunoprecipitated with GFP-Trap-agarose beads. Proteins on the beads were directly digested with Trypsin overnight. Supernatants were collected, acidified to pH2.5 and desalted using GL-Tip SDB followed by LC-MS/MS analysis.

ORGANISM(S): Caenorhabditis Elegans

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD007848 | JPOST Repository | Wed Jan 31 00:00:00 GMT 2018

REPOSITORIES: jPOST

ACCESS DATA

Publications

The autophagy receptor ALLO-1 and the IKKE-1 kinase control clearance of paternal mitochondria in Caenorhabditis elegans.

Sato Miyuki M Sato Katsuya K Tomura Kotone K Kosako Hidetaka H Sato Ken K

Nature cell biology 20171218 1

In Caenorhabditis elegans embryos, paternally provided organelles, including mitochondria, are eliminated by a process of selective autophagy called allophagy, the mechanism by which mitochondrial DNA is inherited maternally. However, it remains unclear how paternal organelles are recognized and targeted for autophagy. Here, we identified an autophagy receptor for allophagy, ALLO-1. ALLO-1 is essential for autophagosome formation around paternal organelles and directly binds to the worm LC3 homo ...[more]

PMID: 29255173

Similar Datasets

Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP). Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4M-BM-0C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 M-NM-<l of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4M-BM-0C. Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells. Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.

Dataset Information

GFP-Trap-IP-phospho

Publications

The autophagy receptor ALLO-1 and the IKKE-1 kinase control clearance of paternal mitochondria in Caenorhabditis elegans.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure