Fig. 8

In vivo evaluation of homo- and hetero-oligomerization of DUF3669 domains of Z746 and Z777. a Analysis of oligomerization between N-terminal fragments of ZNF746 and ZNF777. HEK293 cells that stably express either GST or GST-Z746a/1–279 were transiently transfected with either Z777/1–362 or Z777/1–282 tagged with a One-Strep-Tag (OST). Proteins enriched by either immunoprecipitation with anti-GST rabbit polyclonal antibodies (lanes labeled “IP”) or by pull-down using Strep-Tactin via the OST (lanes “P”) were analyzed by Western blotting alongside an aliquot of the input extract (lanes “X”) with GST and OST antibodies in different color channels (upper and lower panels from the same blot) as indicated. b Narrowing down of the protein oligomerization domains and analysis of homo-oligomerization. HeLa cells were transfected with the indicated GST/GST-fusion and OST-fusion constructs and subjected to Strep-Tactin pulldown via the OST-tagged proteins. Western blots interrogated the input extracts and the enriched fractions (lanes labeled “X” and “P”, respectively) using anti-GST and anti-OST antibodies. Representations of the two-color channels are given here as black/white overlay images. c Alternative analysis of ZNF777 homo-oligomerization using HeLa cells transfected with GST/GST-fusion and GFP/GFP-fusion constructs. Enriched proteins from GST pulldown via glutathione beads (lanes “P”) were probed with anti-GST and anti-GFP antibodies. Images depict black/white representations of the two-color overlays. White block arrows point to GST bands, grey block arrows point to OST bands and black block arrows denote GFP bands