Interaction of endogenous hER with AIB1 is demonstrated by
coimmunoprecipitation. MCF-7 cells were treated with E2 for
0.5 h and extracts prepared as described in Materials and
Methods (CE, lane 1, NEs, lane 2, or
NEv, lane 3). Equal volumes of extract per packed cell
volume were loaded to allow direct visual comparison of lanes.
Immunoprecipitation of NEs (lane 4) or NEv
(lane 5) extracts by using anti-ER antibody was performed and analyzed
by Western blot. (A) Blots were cut in half horizontally
along the 97,000 marker. The top half was immunostained for
AIB1(Mr, 160,000), and the bottom half for
hER (Mr, 66,000). Nonspecific binding of
NEs (lane 6) and NEv (lane 7) extracts is shown
by using anti-IgG immunoprecipitation. The original extracts (lanes 2
and 3) represent 15% of the input into immunoprecipitation reactions.
(B) Experiment conducted as in A, except
the top half was immunostained for SRC-1
(Mr, 160,000). The original extracts (lanes
2 and 3) represent 13% of the input into immunoprecipitation
reactions.