Sf9 cells are routinely maintained at 27°C in tissue culture plates containing Graces medium, 10% FBS, and 10 |g/mL gentamycin. For growth of Sf9 cells in suspension cultures, 0.1% pluronic F-68 is added to the media. Initially, Sf9 cell monolayers are cotransfected with modified baculovirus genomic DNA (Pharmingen, cat. no. 21100D) and either the pVL1392-FLAG-TR or -RXR baculovirus transfer vector construct (see Note 3). The transfection supernatant (containing recombinant f:TR- or f:RXR-expressing baculovirus) is then amplified to a high titer and used to infect Sf9 cells growing in spinner flasks.
Fig. 3. Immunopurified FLAG-tagged nuclear receptors plus associated cofactors can activate transcription in vitro. (A) f:TR-TRAP complex preparation immunopurified from T3-treated HeLa cells stably expressing a f:TRa transgene (a-2 cells; see ref. 5). Immunopurified f:TR-TRAP complex ( 200 ng)(containing approx 20 ng f:TRa) was fractionated on an 8% SDS-polyacrylamide gel and silver-stained. TRAP subunits are indicated on the right. Subunits shown in parentheses indicate suspected human homologs of yeast mediator proteins designated as such on the basis of their size. Lower molecular weight TRAP subunits (<30 kDa) were too small to be resolved on this gel. TR-TRAP preparations containing additional subunits and purified under apparently modified conditions have been reported elsewhere (8) (see Note 15). (B) Baculovirus-expressed FLAG-TRa and -RXRa (bv-f:TRa and bv-f:RXRa, respectively) immunopurified from infected Sf9 cells. Immunopurified bv-f:TRa (100 ng) and bv-f:RXRa (50 ng) were fractionated on an 8% SDS-polyacrylamide gel and stained with Coomassie brilliant blue. (C) Immunopurified bv-f:TR, bv-f:RXR, and the f:TR-TRAP coactivator complex activate transcription in vitro. bv-f:TRa (10 ng) (lanes 1 and 2) or 100 ng of the f:TR-TRAP complex (containing 10 ng f:TRa) (lanes 3 and 4) were added as indicated along with 20 ng of bv-RXRa to a Namalawa cell nuclear extract (40 |lg), and transcription was measured from the TRE3 A53 template (50 ng). The reference template ML200 (25 ng), which lacks TREs, was added to each reaction as an internal control. T3 (10-7 M) was added to the reaction in lane 2. To confirm the involvement of the TRAP coactivator complex in mediating transcriptional activation, 300 ng of affinity-purified polyclonal antibodies against one of the TRAP subunits (a-TRAP220) (20) was added to the reaction in lane 4.
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