μmacs protein g microbeads

Approximately 50 million cells were lysed in Triton® X-100 lysis buffer containing 150 mM NaCl, 50 mM Tris HCl pH 8.0, 1% Triton® X-100 (Plus one, Pharmacia Biotech, Uppsala, Sweden), Complete mini Protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany), 5 mM NaF, 1 mM Na-orthovanadate, 10 mM nicotinamide and 1 μM TSA, and immunoprecipitation was carried out using μMACS ProteinG Microbeads (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer’s procedure. The cell lysate was pre-cleared with μMACS Protein G MicroBeads to remove unspecific binding to the beads followed by a pre-clear using an unspecific antibody (Chromatographically purified Rabbit IgG, Invitrogen, Camarillo, CA, USA) and μMACS Protein G MicroBeads to remove unspecific binding to the immunoglobulines, before new μMACS Protein G MicroBeads and anti-acetyl-lysine antibody (4G12) (Millipore, Billerica, MA, USA) were added to the pre-cleared lysate for immunoprecipitation of acetylated proteins. Proteins were eluted in 95°C SDS loading buffer and loaded directly on to a gel for electrophoresis.

Control- or transfected HEK293T cells (6–8 x 10⁶) were lysed in 1 ml of lysis buffer (PBS/ 1% NP40/ protease inhibitor cocktail), incubated with 1 μg rabbit polyclonal anti-DDX3 antibody (Bethyl Laboratories, Montgomery, USA) at 4°C for 2–3 h, washed extensively with lysis buffer and incubated with 20 μl Protein A/G Plus-Agarose (Santa Cruz Biotechnology) at 4°C overnight. In infected HEK293T cells, DDX3 was immunoprecipitated using the MultiMACS Protein G Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). HEK293T cells (18–20 x 10⁶) were harvested and lysed in 1 ml of μMACS Lysis-Buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl, pH 8) according to the manufacturer’s instruction. Lysates were centrifuged at 13.000 x g, 4°C for 10 min and incubated with 2 μg (20 μl) of mouse monoclonal anti-DDX3 antibody (Santa Cruz Biotechnology) at 4°C for 15 min followed by incubation with 50 μl magnetic μMACS Protein G MicroBeads (Miltenyi Biotec GmbH) for 2 h. Immunoisolation using the μColumns and μMACS Separator were performed according to the manufacturer´s instruction. Immunoprecipitation of FLAG-tagged DDX3 and -PKN was performed with 20 μl Anti-FLAG-sepharose (Anti-FLAG M2 Affinity Gel, Sigma).

Cells were cultured in T175 flasks and at 80% confluency cells were washed once with cold DPBS and then incubated with 2 mM of dithiobis(succinimidyl propionate (Pierce™ Premium Grade DSP, Thermo Fisher Scientific) for 1 h at 4 °C. Then, quenching buffer (1 M Tris, pH 7.5) was added for 15 min at 4 °C. Cells were lysed in 1.2 mL of Pierce ™ IP Lysis Buffer (Thermo Fisher Scientific) containing 1% (v/v) Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher) followed by one wash with cold DPBS. Cell lysates were centrifuged and stored as described above. For the immunoprecipitation 500 µg of protein was incubated with 2–4 µg of primary or nonspecific IgG isotype control antibodies (Supplementary Table S3) overnight at 4 °C on HulaMixer™ Sample Mixer with rotating angle at 45° and speed at 25 rpm (Thermo Fisher). Lysate-antibody complex was then incubated with 100 µL of μMACS Protein G MicroBeads (Miltenyi Biotec) for 1 h at 4 °C using HulaMixer™ Sample Mixer at 25 rpm. Postabsorptive supernatant was magnetically isolated after eluting the lysate-antibody-bead complex through µMACS™ separator and µ column (Miltenyi Biotec) according to the manufacturer's instructions. Epitope location and specificity of the in-house antibodies against sortilin and syndecan-1 used in this method are provided in Supplementary Figs. S12 and S13.