Anti polyhistidine

Rabbit polyclonal antibodies raised against TSEN2 (N-NGDSGKSGGVGDPREPLG-C), TSEN34 (N-QASGEQEEAGPSSSQAGPSNG-C), and TSEN54 (N-RSRSQKLPQRSHGPKDFLPD-C)^{23 (link)} (Gramsch Laboratories, Schwabhausen, Germany) were affinity-purified from rabbit sera and eluted sequentially by addition of 1.5 M MgCl₂ and 0.1 M glycine, pH 2.5. Eluates were dialyzed against HEPES-buffered saline (HBS, pH 7.9) overnight, supplemented with 10% (v/v) glycerol, and stored at −80 °C. Small-scale pilot experiments were set up to assess the suitability of the affinity-purified antibodies for immunoprecipitation experiments using total cell lysate from HEK 293 cells. Antibodies used in western blotting (WB) and immunoprecipitation (IP) were: anti-TSEN15 (rabbit polyclonal, Atlas Antibodies, HPA029237; WB dilution 1:1000), anti-TSEN34 (IP, WB dilution 1:5000), anti-TSEN54 (WB dilution 1:5000), anti-TSEN2 (IP, WB dilution 1:5000), anti-GAPDH (rabbit monoclonal, 14C10, Cell Signaling Technology, #2118; WB dilution 1:1000), anti-β-actin (mouse monoclonal, clone AC-74, Sigma-Aldrich, A2228; WB dilution 1:3000), anti-mouse IgG–peroxidase conjugate (secondary goat polyclonal, Sigma-Aldrich, A2554; WB dilution 1:20.000), anti-rabbit IgG–peroxidase conjugate (secondary goat polyclonal, Sigma-Aldrich, AP307P; WB dilution 1:20.000), anti-polyHistidine (mouse monoclonal, clone HIS-1, Sigma-Aldrich, H1029; IP).

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).

Cells were lysed in MLB that contains protease and phosphatase inhibitors. Cell lysates containing 500 μg of protein were first incubated for 2 h at 4°C with 3.2 μg of anti-MYC (Invitrogen, Carlsbad, CA, USA) or anti-RAS (Upstate Biotechnology) monoclonal antibody and then incubated for 2 h at 4°C with 20 μL of protein G plus/protein A agarose (Calbiochem, Cambridge, MA, USA). Immunoprecipitated complexes were analyzed by Western blotting using an anti-RAS or anti-MYC antibody after washing three times with PBS. For Western blotting, 20 μg of protein were separated on 15% polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After blocking, membranes were incubated for 12 h at 4°C with anti-MYC, anti-RAS, anti-polyhistidine, or anti-actin (Sigma) antibody and then incubated with horseradish peroxidase-conjugated goat anti-mouse antibody at room temperature for 1 h. An ECL kit (Amersham, Bucks, UK) was used to detect the substrate reaction. The relative protein expression was quantified following normalization with the levels of the actin.