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Proteome analysis using mass spectrometry (MS) in acute shYthdf2-mediated knockdown of cultured RGCs

ABSTRACT: To identify mouse retinal proteomics changes after Ythdf2 knockdown, we extracted the proteomics of E15.5 retina neurons which were cultured and infected with lentiviral shYthdf2 or shCtrl by urea. After using 100 mM TEAB (Sigma) to dilute the urea concentration to less than 2 M in each sample, trypsin (Promega) was then added to digest the proteins overnight at 37 °C. Peptides were further desalted by Strata X C18 SPE column (Phenomenex) and labelled with TMT10plex Mass Tag Labelling kit (Thermo Scientific) according to the manufactural instructions. Finally, the labeled peptides were subjected to HPLC fractionation and LC-MS/MS analysis.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Fugui Niu

LAB HEAD: Shengjian Ji

PROVIDER: PXD017775 | Pride | 2022-04-04

REPOSITORIES: Pride

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The m6A reader YTHDF2 is a negative regulator for dendrite development and maintenance of retinal ganglion cells.

Niu Fugui F Han Peng P Zhang Jian J She Yuanchu Y Yang Lixin L Yu Jun J Zhuang Mengru M Tang Kezhen K Shi Yuwei Y Yang Baisheng B Liu Chunqiao C Peng Bo B Ji Sheng-Jian SJ

eLife 20220218

The precise control of growth and maintenance of the retinal ganglion cell (RGC) dendrite arborization is critical for normal visual functions in mammals. However, the underlying mechanisms remain elusive. Here, we find that the N6-methyladenosine (m6A) reader YTHDF2 is highly expressed in the mouse RGCs. Conditional knockout (cKO) of Ythdf2 in the retina leads to increased RGC dendrite branching, resulting in more synapses in the inner plexiform layer. Intere ...[more]

PMID: 35179492

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