Ez view red protein g beads

Immunoprecipitation experiments were realized with 150 μg of total protein amounts from the cytosolic and nuclear fraction in normoxia or hypoxia, with a HA antibody (H9558/H3663, Sigma) 72 μg (2.4 mg/mL) or IgG mouse control (Sigma I5381) (2 mg/mL) using EZ view red protein G beads (Sigma). The beads-antibody-protein mix was incubated overnight at 4 °C and bounds protein were eluted with 35 μg HA peptides diluted in PBS (Sigma); then Laemmli buffer was added and the eluate heated at 95 °C 2 min.

HEK293T cells were transfected with 3xFLAG tagged DAPK1, LRRK1, LRRK2, MASL1, or GFP plasmids using PEI reagent, collected 24 h after transfection and cells were lysed in the buffer: 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton, 10% Glycerol, protease inhibitor cocktail (Roche), and 1x Halt phosphatase inhibitor cocktail (Thermo Scientific). Lysates were precleared by centrifugation at 20 000 × g for 10 min and incubated for 1 h at 4 °C with EZview Red Protein G beads (Sigma) to remove proteins non‐specifically binding to agarose. After preclear with protein G beads, lysates were incubated for 1 h at 4 °C with EZview Red Anti‐FLAG M2 Agarose (Sigma) that is suitable for the immunoprecipitation of FLAG fusion proteins. Beads were washed six times with the wash buffer: 20 mM Tris (pH 7.5), 400 mM NaCl, 1% Triton and proteins were eluted in 25 mM Tris (pH 7.5), 150 mM NaCl, and 100 μg mL⁻¹ 3xFLAG peptide (Sigma). Protein yields and purity were estimated by staining gels with Coomassie brilliant blue staining (Thermo Scientific, Figure 1, Supporting Information).