Cell pellets from synchronized HeLa CCL2 cells and samples from pulldown experiments were resuspended in SDS-sample buffer and briefly denatured at 95°C. Protein was resolved by SDS-PAGE and transferred to nitrocellulose blotting membranes (GE Healthcare). Membranes were blocked over night with 5% skim milk powder in PBS-T (PBS containing 0.1% Tween 20). Subsequently, membranes were incubated at RT for 1 h with indicated antibodies diluted in 5% milk-PBS-T. Primary rabbit polyclonal antibodies directed against NUP188, NUP93, NUP53 and NUP54 have been described (Linder et al., 2017 (link)). Antibodies directed against actin (Sigma, cat no. A1978), HA (Roche), pH3 (Cell Signaling, cat no. 9701S) and NUP62 (Abcam, cat no. ab188413) are commercially available. After three washing steps with TBS-T secondary antibody solutions were applied in 5% milk-PBS-T and membranes kept shaking for 1 h at RT. Subsequent washing was followed by detection. HRP-conjugated secondary antibodies used to detect primary antibodies included goat anti–rabbit IgG and goat anti-mouse IgG (Sigma-Aldrich). Chemiluminescence was initiated using ECL detection reagent (GE Healthcare) and the signal was detected using Fuji RX film (Fujifilm)
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