Luteolin

For Co-Immunoprecipitation (co-IP) of endogenous proteins DYT-PRKRA and wt lymphoblasts were seeded at a concentration of 300,000 cells/ml of RPMI complete media and either left untreated or treated with 50 µM of luteolin (Santa Cruz) for 24 h. When treated with tunicamycin for indicated time periods after luteolin treatment, tunicamycin was added at 5 μg/ml. Cells were harvested at indicated time points and whole cell extract was immunoprecipitated overnight at 4°C on a rotating wheel in IP buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 20% Glycerol) using anti-PKR antibody (71/10, R&D Systems) and protein A sepharose beads (GE Healthcare). Immunoprecipitation was carried out using 100 ng of anti-PKR antibody and 10 µL of protein A sepharose beads slurry per immunoprecipitation. Immunoprecipitates were washed 3 times in 500 µL of IP buffer followed by resuspension and boiling for 5 min in 1X Laemmli buffer (150 mM Tris–HCl pH 6.8, 5% SDS, 5% β-mercaptoethanol, 20% glycerol). Samples were resolved on 10% SDS-PAGE denaturing gel and probed with anti-PACT antibody to determine co-IP efficiency and anti-PKR antibody to determine equal amounts of PKR were immunoprecipitated in each sample. Input blots of whole cell extract without immunoprecipitation are shown to indicate equal amounts of protein in each sample.

Ultrapure water was obtained through a purification system (Millipore Direct-Q UV, Bedford, MA, USA). Formic acid, of LC-MS grade, and ammonium acetate ≥99% were obtained from Fluka (Buchs, Switzerland). Sodium hydroxide >99% and methanol, of LC-MS grade, were acquired from Merck (Darmstadt, Germany). 2-propanol was acquired from Fisher Scientific (Geel, Belgium).
All standards had an analytical grade ≥95%. Epicatechin, ferulic acid, gallic acid, homovanillic acid, oleuropein, pinoresinol, p-coumaric acid, and syringaldehyde were purchased from Sigma-Aldrich (Steinheim, Germany). Apigenin, caffeic acid, tyrosol, and vanillin were acquired from Alfa Aesar (Karlsruhe, Germany). Luteolin and hydroxytyrosol were purchased from Santa Cruz Biotechnologies (Heidelberg, Germany). Syringic acid was obtained from Extrasynthèse (Genay, France). Stock solutions for each analyte (1000 mg/L) were dissolved in methanol and stored at −20 °C. Mixed standard working solutions were prepared every laboratory day diluting the stock solutions with water:methanol (20:80, v/v).

Methanol (LC-MS grade) and sodium hydroxide (>99%) were purchased from Merck (Darmstadt, Germany). Ammonium acetate (≥99% for HPLC) and formic acid (LC-MS Ultra) were purchased from Fluka (Buchs, Switzerland). Isopropanol was obtained from Fisher Scientific (Geel, Belgium). Ultrapure water was provided by a Milli-Q purification apparatus (Millipore Direct-Q UV, Bedford, MA, USA). Regarding the standards that were used, syringic acid 95% was purchased from Extrasynthèse (Genay, France). Gallic acid 98%, ferulic acid 98%, epicatechin 97%, p-coumaric (4-hydroxycinnamic acid) 98%, homovanillic acid 97%, quercetin 98%, oleuropein 98%, pinoresinol 95%, caffeic acid 99%, taxifolin 98%, vanillic acid 97% and syringaldehyde 98% (internal standard) were obtained from Sigma-Aldrich (Steinheim, Germany). Hydroxytyrosol 98% and luteolin 98% were acquired from Santa Cruz Biotechnologies. Vanillin 99%, apigenin (4,5,7-trihydroxyflavone) 97% and tyrosol (2-(4-hydroxyphenyl) ethanol) 98% were purchased from Alfa Aesar (Karlsruhe, Germany). Stock standard solutions of individual compounds (1000 mg/L) were solubilized in methanol and stored at −20 °C in dark brown glass bottles. Intermediate standard working solutions containing the analytes were prepared by appropriate dilution of the stock solutions with methanol:water (80:20, v/v) over the concentration range 0.1–8 mg/L.