C myc human recombinant protein

An analysis of p38α kinase activity was performed using the ADP-Glo Kinase Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The p38α active protein (10 ng, Promega, Madison, WI, USA) was assayed in a kinase reaction buffer with 500 ng c-MYC human recombinant protein (Abcam, Cambridge, MA, USA), 150 µM ATP, and varying concentrations (0.01, 0.1, and 1 µM) of ralimetinib (LY22228820). A total of 500 ng of p38 peptide substrate was used as a control. The generated luminescence was measured using a SPECTROstar Omega microplate reader (BMG Labtech, Offenburg, Germany).

GST-p38α and HIS-β-catenin fusion proteins were generated as previously described [21 (link)]. The GST-p38α human recombinant protein was incubated with c-MYC human recombinant protein (Abcam, Cambridge, MA, USA). The HIS-β-catenin fusion protein was used as a positive control [21 (link)]. Proteins were incubated for 1 h at 4 °C on a rocking platform for in vitro binding. The fusion proteins were precipitated with Pierce Glutathione Magnetic Agarose Beads (Thermo Fisher Scientific by Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions and then washed extensively in buffer A (20 mM Tris-HCl pH 8, 150 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.1% NP-40) containing fresh inhibitors and 1 mM DTT. Afterward, the precipitates were resolved on 10% SDS-PAGE and analyzed by immunoblot. The primary antibodies were anti-polyHistidine (Sigma-Aldrich, St. Louis, MO, USA), anti-GST (Cell Signaling, Danvers, MA, USA), and anti-c-MYC (Cell Signaling, Danvers, MA, USA). Rabbit IgG HRP and Mouse IgG HRP (GE Healthcare, Milwaukee, WI, USA) were used as secondary antibodies and revealed using the ECL-plus chemiluminescence reagent (GE Healthcare, Milwaukee, WI, USA).