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. 2014 Nov 20;5:5531. doi: 10.1038/ncomms6531

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(a) Flag/HA-epitope-tagged NELF-E (eNELF-E) from HeLa S3 Dignam nuclear extracts was sequentially immunopurified on anti-Flag and anti-HA antibody-conjugated agarose beads. Purified material was separated by SDS–PAGE and visualised by silver staining. eNELF-E-associated proteins were identified by MS (see Supplementary Dataset 1). (b) Flag/HA IPs from samples shown in (a) were separated by SDS–PAGE and the presence of eNELF-E-associated proteins identified was confirmed by immunoblotting. (c) Glycerol gradient sedimentation analysis of eNELF-E. Flag-purified eNELF-E-associated complexes were separated by centrifugation through a 12–40% glycerol gradient. Material of even-numbered fractions was resolved by SDS–PAGE and probed for identified proteins. (d) Reciprocal IPs (ReIPs): Flag-purified eNELF-E (Input) was subjected to IP using anti-Spt5, anti-INTS13, anti-HA antibodies or irrelevant rabbit IgG (IPr) or mouse IgG (IPm). Input, IP, as well as flow through (FT) were probed for eNELF-associated proteins.