Fig. 3

a Expression of transiently transfected N-terminally or C-terminally tandem affinity tagged GATA1 detected by anti-GATA-1 immunoblot (top panel), anti-FLAG immunoblot (second panel). Biotinylation of tagged GATA1 was detected by streptavidin–HRP (third panel), whereas hBirA-GFP expression was detected by anti-GFP immunoblot (last panel). The difference in mobility observed between the N-terminally and the C-terminally tagged GATA1 constructs is due to the presence of extra codons that were introduced during cloning of the GATA1 cDNA downstream of the N-terminal tag sequences. The extra bands detected in the anti-FLAG and streptavidin–HRP blots appear to be non-specific as they are not detected by anti-GATA1. b Detection of biotin-tagged GATA1 using anti-GATA1 antibody (upper panel) and streptavidin–HRP (lower panel) in dilutions of nuclear extracts from HEK293 cells transiently transfected with 3xFLAG-TEV-Avi-GATA1/BirA pBUDNeo (lanes labeled as BirA) or with 3xFLAG-TEV-Avi-GATA1/hBirA-GFP pBUDNeo (lanes labeled as hBirA). c Streptavidin pulldowns of nuclear extracts from HEK293 cells transiently transfected with N- or C-terminally tagged GATA-1 constructs. Top panel: detection with anti-FLAG antibody; lower panel: detection of the known GATA1 interacting protein partner ZNF143 co-precipitated with biotinylated GATA1 by streptavidin pulldown. Molecular weight markers (arrows) are in kilodaltons