Pierce anti ha magnetic beads kit

The strains MW001 and MW1104 were grown in Brock medium to OD₆₀₀ = 0.4. Cells were exposed to 100 J/m² UV light (254 nm, Spectroline UV-crosslinker) in 10 ml aliquots and then incubated at 75°C for 3 h. Formaldehyde crosslinking (1% (v/v) final concentration) was carried out in a 35 ml culture for 10 min. The reaction was quenched by addition of 125 mM glycine. After that, cells were harvested by centrifugation at 5000 × g for 20 min (4°C) and stored at −20°C. The cell pellets were resuspended in Pierce IP Lysis/Wash Buffer (0.025 M Tris–HCL pH 7.4, 0.15 M NaCl, 0.001 M ethylenediaminetetraacetic acid, 1% (v/v) NP40, 5% (v/v) glycerol) and then lysed by sonication (40% amplitude, 15-s pulse, 15-s pause) using a KE76 tip (Bandelin). Soluble proteins were obtained by ultracentrifugation at 100 000 × g for 45 min at 4°C. The pulldown assay was performed using the Pierce Anti-HA Magnetic Beads kit (Thermo Scientific) following the manufacturer's instructions. Protein identification was performed by the Mass spectrometry facility of the Center for Biological Systems Analysis (ZBSA) Freiburg.

Coimmunoprecipitation of LC3 with HA-FAM134B or HA-ATL2 was performed in HEK293T cells by transient transfection with 10 µg plasmid for 24 h. Coimmunoprecipitation of ATL3 with HA-ATL2 constructs was performed in HCT116 cells stably expressing the different HA-ATL2 mutant constructs. Immunoprecipitation of all HA-cDNA was performed using Pierce Anti-HA Magnetic Beads Kit (Thermo Fisher) according to the manufacturer’s instructions. Briefly, cells were lysed using the IP-lysis buffer supplemented with Halt protease inhibitor cocktail and Halt phosphatase inhibitor cocktail. Equal amounts of lysates (∼5 mg) with 100 µl HA-magnetic bead slurry were used to perform each immunoprecipitation. Immunoprecipitation was performed at 4°C for 2 h. The beads were washed twice using IP lysis buffer supplemented with 500 mM NaCl, with 5-min incubation on a rotor. The final wash was performed using regular IP lysis buffer. Immunoprecipitated proteins were eluted by boiling the samples at 98°C in 1× NuPAGE LDS sample buffer (Thermo Fisher) for 5 min supplemented with NU-PAGE sample-reducing agent at final 1× concentration.