Anti taf15

PTE, green tea extract (GTE), and black tea extract (BTE) were kindly supplied by the China Academy of Pu-Erh Tea Research [21 (link), 22 (link)]. These extracts were dissolved in distilled water and adjusted to pH 7.4 with 1 M NaOH. Ammonium chloride (NH₄Cl), a lysosomal inhibitor, was purchased from Wako Pure Chemical Industries (Osaka, Japan). Lactacystin (Calbiochem, Bad Soden, Germany) was dissolved in dimethyl sulfoxide (DMSO). Sodium arsenite solution was purchased from EMD Millipore (Darmstadt, Germany). Anti-FUS/TLS, anti-LDLR (low density lipoprotein receptor), anti-TDP-43, anti-V5, anti-β-actin, and horseradish peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Abnova (Taipei City, Taiwan), Cell Signaling Technology (Beverley, MA), Invitrogen (Carlsbad, CA), Sigma (St. Louis, MO), and Thermo Scientific (Waltham, MA), respectively. Anti-EWS and anti-TAF15 were purchased from Cell Signaling Technology (Beverley, MA). The SOD1 antibody was a kind gift from Dr. Noriko Fujiwara.

RIP assay was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore) according to the manufacturers’ instructions. Briefly, immunoglobulin G antibody (Millipore), anti-TAF15 (Cell Signaling Technology), or anti-IGF2BP1 (Abcam) was conjugated to magnetic beads and incubated with the corresponding cell lysates at 4 °C overnight. The coprecipitated RNAs were purified by resuspending beads in TRIzol regent and finally subjected to RT and RT–qPCR.

The ChIP assay was performed using a Pierce Magnetic ChIP Kit (Thermo Fisher Scientific). Cells were fixed with 1% formaldehyde and quenched with glycine, respectively. Nuclei were harvested and sonicated to generate DNA fragments of ∼200 bp. The sonicated chromatin was immunoprecipitated with anti-TAF15 (Cell Signaling Technology) antibody. Immunoprecipitated DNA was purified with spin columns and then analyzed by RT–qPCR. The primer sequences are listed in Table S8.
ChIP-Seq was conducted by Lc-bio Technologies. The high-throughput DNA sequencing libraries were prepared using VAHTS Universal DNA Library Prep Kit for Illumina V3 (catalog no.: ND607; Vazyme). The library products corresponding to 200 to 500 bps were enriched, quantified, and finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model. Raw sequencing data were filtered by Trimmomatic (version 0.36), and clean data were mapped to reference genome using STAR (version 2.5.3a). Read distribution analysis, peak calling, and peak annotation/peak distribution analysis were performed by RSeQC (version 2.6), MACS2 (version 2.1.1), and bedtools (version 2.25.0), respectively. The Homer (version 4.10) was used for motif analysis. All packages were open sources from python software.