llin/streptomycin. All cells were maintained at 37uC with 5% carbon dioxide. Plasmid constructs were introduced into cells as CaPO4 precipitates. Immunocomplexing assays Cell monolayers were TAK 438 free base web washed three times with ice cold phosphate buffered saline. Immunoprecipitation lysis buffer containing protease inhibitors was added directly to the cell monolayer. Cell monolayers were incubated with lysis buffer, lysates were collected and centrifuged to remove cellular debris. Nuclear extracts were generated as described by Schreiber et al.,. Briefly, PBS rinsed cell monolayers collected by centrifugation, were resuspended and incubated 
for 15 min in a hypotonic lysis buffer containing protease inhibitors. Following the addition of NP-40 cell suspensions were centrifuged, and nuclear pellets were resuspended in nuclear extract buffer containing protease inhibitors. Following a 15 min incubation with repeated vortexing, insoluble materials were removed by centrifugation. Supernates were adjusted with hypotonic lysis buffer to a final salt concentration of 125 mM. Supernates were incubated with protein-A sepharose, centrifuged to pellet the sepharose beads and supernates were incubated with protein-A sepharose beads and the indicated antibody overnight. Materials bound to the protein-A sepharose beads were collected by centrifugation, supernates were removed and pelleted materials were subject to four cycles of resuspension /centrifugation. Following removal of the final supernate Laemmli buffer was added to the pelleted materials. Denatured samples were subjected to Western analysis as described previously. All immunocomplexing assays were repeated at least three times. Materials and Methods Plasmids, antibodies and growth factors Flag-tagged SIMPL, amino acids 1-259 of the mouse SIMPL protein, was expressed in the pFLAG-CMV-2TM expression vector. SIMPL antibody, generated by immunizing rabbits against full length recombinant SIMPL protein, was affinity purified against the recombinant protein to enrich for SIMPL specific antibody. 23237488 The p65 antibody was from Millipore; MED1 antibody was from Abcam; antibody to RNAPIIa and to RNAPIIo were from Covance. Anti-FLAGH M2 monoclonal antibody and b-actin antibody were obtained from Sigma Aldrich. Goat anti-mouse IgG F2 fragment specific-HRP was obtained from Jackson ImmunoResearch Laboratories, Inc.. Recombinant human TNFa and recombinant mouse SCF were from PeproTech Inc.; recombinant human erythropoietin; pokeweed mitogen spleen cell conditioned media was from StemCell Technologies. Generation and analysis of TAP-tagged SIMPL The wild-type SIMPL coding sequence was subcloned into the pNTAP-A vector. The TAP-tagged wild-type SIMPL construct was sequenced, expressed in HEK 293 cells to confirm production of the correctly sized fusion protein and to confirm that the SIMPL fusion protein was capable of inducing the activity of an NF-kB 15210823 dependent reporter when overexpressed. To generate TAP-SIMPL containing protein complexes, HEK 293 cells were transfected with empty vector or the TAP-tagged SIMPL construct, twenty-four hours later cell monolayers were rinsed and harvested in PBS. Whole cell lysates were generated SIMPL Modulates TNFa Dependent Trancription using the InterPlayTM lysis buffer followed by three successive freeze/thaw cycles. Lysates were incubated with a streptavidin resin to enrich for TAP-SIMPL containing complexes. The streptavidin resin was washed, and bound complexes were eluted from the
