Co-Immunoprecipitation

The protocol was adapted from a procedure described previously (Gurnett et al., 1997).

Briefly, a tsA-201 cell pellet derived from one confluent 75 cm² flask was resuspended in co-IP buffer (20 mM HEPES (pH 7.4), 300 mM NaCl, 1% Digitonin and PI), sonicated for 8 s at 20 kHz and rotated for 1 hr at 4°C. The samples were then diluted with an equal volume of 20 mM HEPES (pH 7.4), 300 mM NaCl with PI (to 0.5% final concentration of Digitonin), mixed by pipetting and centrifuged at 20,000 x g for 20 min. The supernatants were collected and assayed for total protein (Bradford assay; Biorad). 1 mg of total protein was adjusted to 2 mg/ml with co-IP buffer and incubated overnight at 4°C with anti-GFP polyclonal antibody (1:200; BD Biosciences). 30 μl A/G PLUS Agarose slurry (Santa Cruz) was added to each tube and further rotated for 2 hr at 4°C. The beads were then washed three times with co-IP buffer containing 0.2% Digitonin and deglycosylated as previously described alongside with aliquots of the initial WCL prior to co-IP. Laemmli buffer with 100 mM DTT was added to 1 x final concentration and samples were analysed by SDS-PAGE and western blotting with the indicated antibodies as described previously (Kadurin et al., 2016).

The human embryonic kidney tsA-201 cells were obtained from the European Collection of Authenticated Cell Cultures (# 96121229) and tested to be mycoplasma-free.