RACK1 interacts with PP1cδ via TIMAP. (A): Bacterially expressed glutathione S-transferase (GST) and GST-tagged wild-type TIMAP were loaded onto glutathione-Sepharose as described in Materials and Methods. After a washing step the resin samples were incubated with BPAEC lysate (CL) or cell lysis buffer (LB). Non-binding proteins were washed out and the bound proteins were eluted with 10 mM glutathion. Western blot probed with RACK1 specific antibody (A) of the endothelial cell lysate (CL) and the eluted fractions after the pull-down are shown. (B,C): RACK1 or TIMAP was immunoprecipitated from lysates of BPAEC (B) and BPAEC or HeLa (C) cells as described in Materials and Methods. IP complexes were probed for TIMAP and RACK1 (B) or PP1cδ (C). CL: cell lysate, Ø AB: control of IP from BPAEC without the addition of antibody. (D,E): RACK1 or GFP was immunoprecipitated from non transfected (CL), non-siRNA, TIMAP specific siRNA (si TIMAP), pEGFP-C1 (GFP), pEGFP-C1 TIMAP WT (GFP-Twt) or pEGFP-C1 TIMAPΔpp1c (GFP-TΔ) transfected HeLa cell lysates. IP complexes were probed for GFP, RACK1 or PP1cδ.