Isolation of GATA1-containing complexes in L8057 megakaryocyte cells. (A) Scheme of in vivo protein biotinylation and purification by streptavidin beads. (B) Nuclear extracts from wild-type (UnT), BirA-expressing (BirA), biotag-GATA1–expressing (+ ve), and 6 independent L8057 clones expressing biotag GATA1 and BirA biotin ligase were tested by Western blot with anti-GATA1 antibody (top panel). The top band corresponds to the slower migrating biotag GATA1; bottom band, endogenous GATA1. The blot was probed with antistreptavidin-HRP antibody, which confirms biotinylation of GATA1 in extracts from clones 3, 4, 5, and 6. (C) Crude nuclear extracts from cells transfected with either BirA alone (BirA) or BirA/biotag GATA1 (BirA/biotag GATA1) were incubated with streptavidin-coated beads. Precipitated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (PD lane) and stained with Coomassie blue. Approximately 20 μg crude extract was loaded as input (In lane). indicates biotinylated GATA1 (biotag GATA1), as determined by mass spectrometry. (D) Table of proteins and the number of peptides precipitated by streptavidin beads and identified by mass spectrometry. (E) Gel filtration analysis (top). An example of fractionation of crude nuclear extracts from L8057 cells transfected with BirA/biotag GATA1 on a Superose 6 column. Similar results were obtained from wild-type nuclear extracts. indicates position where protein molecular markers elute. The UV profile indicates that proteins elute in a broad fractionation profile. Fractions were taken from the Superose 6 column, precipitated, and analyzed by Western blotting for GATA1 and several potential protein partners (bottom panels). The antibody used is indicated on the lefthand side of the panel. Note that endogenous GATA1 and biotag-GATA1 have a similar elution profile. Vertical line(s) have been inserted to indicate a repositioned gel lane.