Anti actin

Antibodies used were anti-RIPK1 (BD Biosciences, San Jose, CA, USA, #610459), anti-actin (MP Biomedicals, Solon, OH, USA, #69100), anti-IκB-α (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-371), anti-hRIPK3 (ThermoFisher Scientific Pierce, Waltham, MA, USA, PA1-41533), anti-mRIPK3 (Sigma-Aldrich, St Louis, MO, USA, #R4277 and IMGENEX, San Diego, CA, USA,, IMG-5523-2), anti-hMLKL (Genetex, Irvine, CA, USA, GTX107538), anti-mMLKL (Millipore, Billerica, MA, USA, #MABC604 and Abgent, San Diego, CA, USA, AP14272b-ev), anti-phospho-hMLKL (Abcam, Milton, Cambridge, UK, #187091), anti-hFADD (BD Biosciences, 610399), anti-mFADD (Millipore, 05-486), anti-thiophosphate ester (Epitomics, Burlingame, CA, USA, #2686-1), anti-Braf (ThermoFisher scientific, MA5-15495), anti-HPK1 (Cell Signaling, Danvers, MA, USA, #4472), anti-Hsp90 (Santa Cruz Biotechnology, sc-7947), anti-p38MAPK (Cell Signaling, #9212), anti-ERK1/2 (Cell Signaling, #9102), anti-Flag-HRP (Sigma-Aldrich, St Louis, MO, USA, #A8592) and anti-tubulin-HRP (Abcam, #ab21058). Secondary antibodies used were HRP-conjugated secondary antibodies against mouse, rabbit or rat immunoglobulin (GE Healthcare, Little Chalfont, Amersham, UK).

Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (Roche). Protein quantification was performed with the BCA protein assay kit (Thermo Fisher Scientific). 25 μg protein extracts were loaded on 10% acrylamide/bis acrylamide SDS page gel. After electrophoresis, protein transfer was performed onto a nitrocellulose membrane (GE Healthcare). Membranes were stained with primary antibodies overnight at 4°C and secondary HRP conjugated antibodies (Santa Cruz Biotechnology) for 1 hour RT. Antibodies used were: anti-ADAR1 [#ab168809, Abcam (Figures 1, 2) and #14175, Cell Signaling Technology (Figure 3)], anti-STAT1 and anti-STAT2 (#14995 and #72604, Cell Signaling Technology) and as a loading control anti-actin (#0869100, MP Biomedicals) or anti-tubulin.

For western blot analysis, the cellular extracts were solubilized in 3× Laemmli buffer consisting of 1% SDS and boiled for 5 min at 95 °C. Equal protein amounts (30–80 μg) were separated on SDS–polyacrylamide gel electrophoresis (12.5–15% gels) and transferred to nitrocellulose membranes. Antibody detection was accomplished using the enhanced chemiluminescence method (Thermo Fisher Scientific, Darmstadt, Germany) and developed either with the Fusion SL Imager (Vilber Lourmat, Eberhardzell, Germany) or the Curix60 processor (Agfa healthcare, Bonn, Germany). The following antibodies were used in 3% milk/TBS–Tween (0.1%): anti-mFas (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-actin (1:40,000, MP Biomedicals, Eschwege, Germany), anti-tubulin (Bio-Rad, Munich, Germany), anti-E-cadherin (BD Transduction laboratories, Heidelberg, Germany), anti-hFas, anti-p65, anti-phospho-p65, anti-IκBα, anti-phospho-IκBα, anti-cleaved caspase-3 and anti-Bcl-x_L (Cell Signaling, Leiden, The Netherlands).

Western blotting was performed as previously described [27 ]. The following primary antibodies were used: anti-FASN (1:1000, BD Bioscience, San Jose, CA); anti-TNF-α, anti-IL-1β, anti-IL-6 and anti-NFκB (all 1:500, Santa Cruz Biotechnology, Santa Cruz, CA); anti-phosphoNMDAR2B (Tyr1472) (1:2000, Affinity Bioreagents, Golden, CO) and anti-NMDAR2B (1:1000, Chemicon, Temecula, CA). Loading control was performed by reprobing the membranes with anti-actin (1:10,000; MP Biochemicals, Aurora, OH), anti-NeuN (1:1,000; Chemicon) or anti-N-cadherin (1:1000, BD Bioscience). Membranes were incubated with the corresponding horseradish peroxidase-conjugated antibody (1:2,000; Promega, Madison, WI). Immunoreactive bands were visualized using the Western Blotting Luminol Reagent and quantified by a computer-assisted densitometer (Gel-Pro Analyzer, version 4, Media Cybernetics).

Immunoblotting was carried out as previously described [55 ]. The following antibodies were used in this study: anti-PARP-1, anti-cleaved caspase-3, anti-cleaved caspase-8, anti-caspase-9, anti-cleaved caspase-9, anti-HO-1, anti-NOXA, anti-CHOP, anti-GRP78, anti-PUMA (Cell signaling Technology, Beverly, MA), anti-actin (MP Biomedicals, Solon, OH), goat anti-rabbit IgG-horseradish peroxidase (HRP), and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Plasmids were introduced into protoplasts by polyethylene glycol-mediated transformation⁴⁷ . Protein extracts were prepared at 24 h after transformation or at the indicated time points⁴⁸ . The protoplasts were treated with tunicamycin (10 μg/mL; Sigma-Aldrich, St. Louis, MO) immediately after transformation. Cycloheximide (50 μg/mL; Sigma-Aldrich, St. Louis, MO) treatment was applied at 12 h after transformation. Protein extracts were analyzed by western blotting with anti-HA (Roche Diagnostics, Indianapolis, IN), anti-actin (MP Biomedicals, Solon, OH), anti-GFP (Bio-Application, Pohang, Korea), or anti-BiP antibodies^{47 –49} . The protein blots were developed with an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ), and images were obtained using an LAS4000 image analyzer (Fujifilm, Tokyo, Japan). To quantify expression levels, intensity of protein bands in western blot images was measured using Multi-Gauge program equipped to LAS4000 image analyzer (FUJIFILM, Tokyo, Japan). Band intensities were added in cases of glycosylated proteins except the bottom band that was considered as the non-glycosylated form.