Flag tag and GFP-scFv fusion were obtained by cloning HindIII-XbaI the antibody cDNA in the pcDNA3.1 and in the pEGFP-C1 (Clontech) vectors, respectively. Supplementary Fig. S1.   scFv specificity for NPMc+. (A) ELISA test. Absorbance values were measured at 405 nm using scFv-expressing bacterial supernatants in combination with either NPMc+-MBP fusion fragment or MBP alone. (B) Sequence of the VH and VL domains of the selected anti-NPMc+ scFv antibody. (C) The protein fractions

corresponding to the purified constructs of the NPMc+ fragment 255–298 fused to either MBP (NPMc+ fragment-MBP, lane 2) or the full-length NPMc+ mutant fused to GST (NPMc+-GST, lane 6), were separated by SDS-PAGE Selleck Alectinib gel in parallel with purified MBP (lane 1), GST (lane 5), and the total lysate recovered from either non-transfected insect cells (lane 3) or insect cells expressing GFP (lane 4), the mutant NPMc+ (lane 7), and the wild type NPM1 (lane 8). Protein bands were identified by western immuno-blot using scFv-containing cell culture supernatant in combination

with mouse anti-Myc monoclonal antibody (9E10). (D) Insect cell lysates expressing either NPMc+ (lane 1) or wild type NPM1 (lane 2) were separated by SDS-PAGE gel and probed with mouse monoclonal antibodies specific for either the wild type NPM1 C-terminal end (338) or for the common N-terminal region of NPM (376). HeLa cells were grown in Dulbecco’s modified Eagle Medium (Lonza) supplemented with 10% FBS, l-glutamine (2 mM), penicillin (100 U mL−1), streptomycin (100 mg mL−1). 5-Fluoracil cost OCI-AML2 and OCI-AML3 [29] cell lines were grown in MEM Alpha + GlutaMAX™-I medium Verteporfin manufacturer (Gibco) supplemented with 20% FBS, glutamine and antibiotics. Transient transfections were performed using Lipofectamine™ 2000 (Invitrogen). Sf9 (Spodoptera frugiperda) insect cells were cultured at 27 °C in Sf 900 II SMF medium (Gibco) and transfected with pFastBacDual

plasmids (Invitrogen) expressing either wild type NPM1 or NPMc+ using Insectogene T030-1.0 (Biontex). Baculoviral supernatant was collected after 96 h and used for two cycles of infection. For immunoprecipitation, cells were lysed in 50 mM Tris–HCl, pH 8, 150 mM NaCl, 0.5% NP40, and protease inhibitors. Ten micrograms of scFv were added overnight at 4 °C to HeLa and OCI-AML3 cell lysates followed by protein A/G-sepharose (GE Healthcare). For co-immunoprecipitation experiments, total cell lysate was incubated with mouse M2 anti-Flag agarose beads (Sigma) and with anti-mouse IgG agarose beads (Sigma) for 4 h at 4 °C. Precipitated recombinant purified proteins and cell lysates were separated by SDS-PAGE gel and immunoblotted over a nitrocellulose membrane (Whatman). After incubation with primary antibodies in 5% skimmed milk, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (Bio-Rad).