Hnrnpk

For immunocytochemistry, cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT). Cells were permeabilised and nonspecific antibody binding was blocked using 0.3% Triton-X containing 5% bovine serum albumin (BSA) (Sigma) in PBS for 60 min. Primary antibodies used were SMI-32 (BioLegend, San Diego, CA, USA; 801,701; mouse; 1:1000), ChAT (Millipore, Burlington, MA, USA; AB144P; goat; 1:100), β-III-tubulin (abcam, Cambridge, UK; ab41489; chicken; 1:1000), TDP-43 (ProteinTech, Rosemont, IL, USA; 12892-1-AP; rabbit; 1:400), SFPQ (abcam, Cambridge, UK; ab11825; mouse; 1:400), FUS (Santa Cruz, Santa Cruz, CA, USA; sc-47711; mouse; 1:200), hnRNPA1 (Cell Signaling, Danvers, MA, USA; 8443S; rabbit; 1:500) and hnRNPK (Santa Cruz, Santa Cruz, CA, USA; sc-28380; mouse; 1:500). Primary antibodies were made up in 5% BSA and then applied overnight at 4 °C. Cells were washed with PBS 2 times, and secondary antibody was added using a species-specific Alexa Fluor-conjugated secondary antibody (Life Technologies, Carlsbad, CA, US) at 1:1000 dilution in 5% BSA for 90 min at RT in the dark. Cells were washed once in PBS containing 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (1:1000) for 10 min. Following a second wash in PBS, cells were left in PBS in the dark.

Whole cell extracts were isolated using 1× passive lysis buffer (Promega) and protease inhibitors (Sigma-Aldrich). Total proteins were quantified using Bradford Protein Assay (Bradford Reagent, Bio-Rad). For western blot analysis, the proteins were separated by SDS-PAGE under reducing conditions, transferred on PVDF membranes (Merk Millipore) and incubated with the following primary antibodies: anti-Flag (Sigma-Aldrich), anti-Dnmt3a (Cell Signalling Technology and Santa Cruz), anti-Lin28a (Abcam and Cell Signalling Technology), anti-Gapdh, Ddx3x, Ddx5, Ddh9, Hnrnph1, Hnrnpk, Hnrnpu (all from Santa Cruz Biotechnology), anti-Tut-4, anti-Tut-7 and anti-Syncrip (all from Proteintech).

Mass spectrometry-identified proteins were confirmed by Western blot. Jurkat nuclear extracts after DNA pulldown assay were lysed in sample buffer [62.5 mM Tris·HCl (pH 6.8 at 25°C), 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% wt/vol bromophenol blue]. Equal amounts of protein were loaded onto a 10% SDS-PAGE gel (GTX gel BioRad USA). After it resolved, samples were blotted to Nitrocellulose paper using the Trans-blot Turbo Transfer System (BioRad, USA). Membranes were blocked using LI-COR blocking buffer for 2 h and then incubated with primary antibody 1:1,000 dilution (hnRNP-K, Santa Cruz USA) at 4°C overnight, and with a donkey anti-mouse IR-Dye 800 (LI-COR, USA) secondary antibody for 1 h. The membrane was imaged with a LI-COR Odyssey using Auto-Scan. Background-subtracted signal intensity was quantified using Image Studio 4.0 software.

Equal amounts of protein were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Antibodies and dilutions used in this study were as follows: 1:2,000 AIP1/ALIX (Millipore), 1:4,000 RIPK1, 1:7,500 hnRNP K (Santa Cruz), 1:1,000 PFAS (abcam), 1:2,000 ACYL (Millipore), 1:1,000 VDP p115/p115 (Novus Biologicals), 1:2,000 p115 (abcam), 1:1,000 GBF1 (abcam), 1:500 α-tubulin (Santa Cruz), 1:3,000 G3BP1 (BD Transduction Science), 1:3,000 VP1 (Dako), 1:2,000 Flag (Sigma-Aldrich), and 1:1,000 PARP (Santa Cruz).

BMDMs were harvested and lysed in RIPA buffer supplemented with protease inhibitor (P8340, Sigma-Aldrich, US). Protein concentrations in cell lysates were measured using Pierce™ BCA protein assay kit (23225, Thermo Fischer Scientific, US), and the same amount of protein was loaded and separated on a polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane. Membranes were blocked with 5% BSA solution (prepared in TBST) for 2 hours and probed with primary antibodies overnight in dilution buffer (TBST supplements with 1% BSA). The antibodies used in Western blots are: hnRNPA2/B1 (Santa cruz, sc-32316), hnRNPL (Abcam, ab6106), hnRNPK (Santa cruz, sc-28380), Cxadr (Santa cruz, sc-373791) and Gapdh (Santa Cruz, sc-365062). Membranes were probed with horseradish peroxidase-conjugated anti-mouse and anti-goat secondary antibodies (sc-2005 and sc-2020). Western blots were developed with LumiGlo Reserve™ chemiluminescent substrate kit (54-64-01, Sera care, Life Sciences, MA, US).