Hnrnp l

For Western blot analysis, equal amounts of protein (adjusted from Coomassie stains) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Whatman). After blocking with 5% (w/v) low-fat milk in TBS/0,1%/Tween-20 (Roth), quantitative immunoblot analyses were performed using primary antibodies against hnRNP K (polyclonal, anti-rabbit, 1:500, Abcam), hnRNP L (monoclonal, anti-mouse, 1:2000, Abcam), α-synuclein (monoclonal, anti-mouse, 1:1000, BD Biosciences), NEFL (monoclonal, anti-rabbit, 1:50000, Novus), and β-actin (monoclonal, anti-mouse, 1:4000, Sigma), followed by appropriate HRP-coupled secondary antibodies in 5% low-fat milk/TBS/0,1% Tween-20. Immunoreactive bands were detected using HRP-chemiluminescence substrate (Pierce ECL Western Blotting Substrate, Thermo Scientific) and exposition on Amersham Hyperfilm (GE Healthcare). Signals on the developed films were digitised (GS-800, BioRad) and quantified using QuantityOne v4.6.9 (BioRad). Signals were normalised to Coomassie stains because the present proteome analyses have revealed that the abundance of proteins commonly used for normalisation (e.g., GAPDH and cytoskeletal components, such as tubulin and actin isoforms) also showed changes in response to drug treatment (cf. Additional file 1: Table S1).

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).

C33A cells were fixed with 1% formaldehyde for 10 min at room temperature and chromatin extracted as described in Ryme et al. (2009 (link)). For RNA regulatory protein 1.5–2% formaldehyde was used (Görnemann et al. 2005 (link)). The chromatin was fragmented by sonication to fragments with a mean length of 500 bp. The antibodies used: BRM, SAM68, hnRNPL, hnRNPU, BAF155/SMARCC1, BAF250/ARID1, BAF200/ARID2, and BAF180/PBRM1 were purchased from Abcam, BAF200/ARID2 and BAF180/PBRM1 were from Bethyl Laboratories Inc and BRD9 were from Cell signalling and Anova (Supplementary Table S6). Primers used in the analysis are presented in Supplementary Table S5. The standard was SD, and p value calculated according to Student’s t test.