Sc 350

Co-immunoprecipitation (Co-IP) of proteins as an indication of protein-protein interactions was carried out as described earlier [34 (link), 35 (link)]. Briefly, cells were washed with 1X PBS and harvested in NP-40 Buffer (50mM Tris pH 7.5, 150mM NaCl, 2mM EDTA, 0.5% NP-40 supplemented with PMSF and protease inhibitors). Cells were lysed for 30min on ice and passaged through a 27G needle three times. Lysates were centrifuged and protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific; Waltham, MA). Equal protein amounts were used for IP. Protein extracts were precleared with protein A agarose rocking at 4°C for one hour. The extract/bead mix was centrifuged and the supernatant was transferred to new tubes. Extracts were then incubated with an antibody against p53 (PAb 421), CBP (sc-369, Santa Cruz), Sp1 (sc-59, Santa Cruz), or Ets1 (sc-350, Santa Cruz) and protein A agarose beads while rocking at 4°C overnight. The following morning the extract/bead/antibody mix was centrifuged and the beads were washed three times with NP-40 Buffer. The buffer was removed and equal volume 2X Laemmli loading buffer was added and boiled for ten minutes. Extracts were then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, a small aliquot of the IP supernatant was set aside and co-electrophoresed as a loading control.