then, the protein - rna complexes were isolated, and the rnas were extracted by the phenol - chloroform rna extraction method. the relative interaction between the target proteins and rnas was analyzed by qpcr with normalization against the corresponding input. endosomes were isolated using the minute™ endosome isolation and cell fractionation kit ( invent biotech, plymouth, mn ), according to the manufacturer ’ s instructions. briefly, the final isolated pellets were lysed with 1x cell lysis buffer ( cat # 9803, cell signaling, danvers, ma ) containing invitrogen™ superase • in™ rnase inhibitor ( invitrogen, carlsbad, ca ). for rna isolation from endosomes, trizol or an rna clean & concentrator - 5 kit ( zymo research, irvine, ca ) was used according to the manufacturer ’ s instructions. the relative levels of target rnas in endosomes were normalized against hprt1 mrna, 18s rrna, or the total u6 snrna in the input samples. the nuclear, cytoplasmic, or total rnas were isolated using the rna subcellular isolation kit ( active motif, carlsbad, ca ) according to the manufacturer ’ s instructions. the relative rna levels were measured by qpcr analysis. the nuclear and cytoplasmic proteins were isolated using the ne - per™ nuclear and cytoplasmic extraction reagents kit ( thermo scientific, 78833 ) according to the manufacturer ’ s instructions, followed by immunoblotting analysis. trna ( sigma - aldrich, st. louis, mo ), u6 ( idt, coralville, ia ), ma43 u6 ( idt, coralville, ia ), or poly i: c ( sigma - aldrich, st. louis, mo ) were labeled with an rna 3 ’ end biotinylation kit ( thermo fisher scientific, waltham, ma ). the quality and quantity of the biotin - labeled rnas were confirmed using the chemiluminescent nucleic acid detection module kit ( thermo fisher scientific, waltham, ma ) according to the manufacturer ’ s instructions. streptavidin - coated magnetic beads, dynabeads™ myone™ streptavidin c1 ( thermo fisher scientific, waltham, ma ), were incubated with the prepared ip samples, including biotin - labeled target rnas with or without recombinant proteins such as human ythdf2 ( active motif, carlsbad, ca ) or human tlr3 ( abcam, waltham, ma ). 1x cell lysis buffer ( cat # 9803, cell signaling, danvers, ma ) was used as an ip reaction buffer for cell lysates or recombinant proteins. the relative binding affinity between biotin - labeled rnas and target proteins was measured by immunoblotting. all experiments were conducted using established cell lines and immunoprecipitation assays; ethical approval was not applicable. to characterize proteins associated with ythdf2, immunoprecipitate s were analyzed by mass spectrometry. a total of three samples were submitted: one igg control and two ythdf2 immunoprecipitate s ( treated and non - treated ). sample preparation, lc – ms / ms acquisition, and data processing were performed by ms bioworks ( ms bioworks llc, ann arbor, mi ). for sample preparation, half of each immunoprecipitate was subjected to sds - page using a 10 % bis - tris nupage gel ( invitrogen ) with the mes buffer system. gels were run to ~ 2 cm, and the corresponding region was excised into ten equal slices. gel slices were processed for in - gel digestion on a progest robot ( digilab ) using the following protocol: washing with 25 mm ammonium bicarbonate and acetonitrile, reduction with 10 mm dithioth reitol at 60 °c, alkylation with 50 mm iodoacetamide at room temperature, digestion with sequencing - grade trypsin ( promega ) at 37 °c for 4 h, and quenching with formic acid. supernatants were analyzed directly without further cleanup. half of each digested sample was analyzed by nanol c - ms / ms using a waters m - class hplc system coupled to a thermo fisher orbitrap fusion lumos mass spectrometer. peptides were first loaded onto a trapping column and then separated on a 75 μm analytical column packed with luna c18 resin ( phenomenex ) at 350 nl / min. elution was performed with a linear gra dien t of acetonitrile in 0. 1 % formic acid. the mass spectrometer was operated in data - dependent acquisition mode, with full ms scans acquired at 60, 000 fwhm and ms / ms scans at 15, 000 fwhm, using a 3 s cycle time. data analysis was performed using mascot ( matrix science ) against the swissprot human database ( concatenated forward and reverse sequences plus common contaminants ). search parameters were as follows: enzyme specificity set to trypsin / p with up to two missed cleavages; carbamidomethylation of cysteine as a fixed modification; oxidation of methionine, n - terminal acetylation, pyro - glutamate formation ( n - terminal glutamine ), deamidation ( n, q ), and phosphorylation ( s, t, y ) as variable modifications; monoisotopic mass values; precursor ion tolerance of 10 ppm; and fragment ion tolerance of 0. 02 da. mascot dat files were parsed into scaffold ( proteome software ) for validation and filtering. data were filtered at 1 % peptide and protein false discovery rate ( fdr ), requiring at least two unique peptides per protein the anti - ythdf2 ps39 antibod y was generated by abclonal technology. one - and - a - half - year - old new zealand rabbits ( 2. 5 kg ), housed under spf conditions, were subcutaneously injected with 700 μg of a modified antigen peptide ( epyl ( s - p ) pqar - c - klh ) emulsified with complete freund ’ s adjuvant ( cfa ) for the primary immunization, followed by five booster injections of 350 μg of the same peptide emulsified with incomplete freund ’ s adjuvant ( ifa ) at 1 -, 2 -, and 3 - week intervals. terminal bleeds were collected after the final immunization. polyclonal antibod ies were purified from the terminal bleeds by antigen affinity chromatography using a column conjugated with the modified peptide ( epyl ( s - p ) pqar - c ), followed by depletion using a column conjugated with the non - modified peptide ( epylspqar - c ). gene ontology ( go ) enrichment and kyoto encyclopedia of genes and genomes ( kegg ) pathway enrichment analyses were performed using metascape ( https: / / metascape. org / ), a web - based portal designed to provide a comprehensive gene list annotation and analysis resource, as described previously, using the default background gene list ( all genes ) provided by metascape for initial pathway screening. quantitative venn diag rams were generated using the following web tool: https: / / www. deepvenn. com /. genes with an adjusted p value less than 0. 05 by deseq2 were used for pathway analysis and venn diag ram analysis. migration assays were performed as described previously. briefly, 5 × 10 cells were suspended in 150 μl of serum - free medium and seeded onto 8 - mm pore transwell inserts ( corning, corning, ny ) for the migration assay. the lower chamber was filled with 900 μl of complete medium. cells on the transwell inserts were then fixed with 4 % paraformaldehyde / pbs for 30 min. subsequently, fixed cells were stained with hematoxylin solution ( sigma - aldrich, st. louis, mo ) for 1 h. after wiping off cells on the upper side of the filter on the transwell inserts using cotton swabs, microphotograms of the cells migrated onto the lower side of the filter were taken using light microscopy. cells migrated onto the lower side of the filter were manually counted from the microphotograms. mean cell numbers were quantified from about ten randomly selected squares ( 500 μm x 500 μm ) per transwell insert. cell proliferation was assessed using the cell counting kit - 8 ( cck - 8; sigma - aldrich, st. louis, mo ) according to the manufacturer ’ s instructions, with minor modifications. briefly, cells were seeded in 24 - well plates and incubated with cck - 8 solution for 1 h. after incubation, the culture medium containing the cck - 8 reagent was transferred to 96 - well plates, and absorbance was measured at 450 nm using a microplate reader.