Anti flag antibody

HEK293T cells were co-transfected with HA-tagged ubiquitin and other indicated plasmids for 42 h and cells were added with MG132 at final concentration of 20 μM for 6 h, then cells were collected and lysed. The samples were incubated with anti-Flag antibody in addition with protein G-Sepharose (GE Healthcare) and separated by SDS-PAGE and analyzed by western blotting.

NPM-ALK autophosphorylation was measured by modified methods as described previously [16 (link)]. Cells were lysed in NP-40 lysis buffer supplemented with protease inhibitors and NPM-ALK was immunoprecipitated using the anti-Flag antibody and protein G-sepharose (GE Healthcare Biosciences, Pittsburgh, PA, USA) at 4°C for 4 hr. Immunocomplexes were washed three times with NP-40 lysis buffer and twice with kinase buffer (20 mM Tris-HCl pH 7.4, 5 mM MgCl₂, 5 mM MnCl₂, and 1 mM dithiothreitol). The kinase reaction was performed in 30 μL of kinase buffer including 30 μM ATP (Cytoskeleton, Inc., Denver, CO, USA) and 10 μCi [γ-³²P] ATP (PerkinElmer, Inc. Waltham, MA, USA) at 30°C for 15 min. The reaction was terminated by the addition of Laemmli buffer and further boiling for 10 min. Proteins were separated by SDS-PAGE and ³²P-labeled proteins were measured by autoradiography. To show the relative phosphorylation level of NPM-ALK, the band intensity of phosphorylated NPM-ALK was normalized with expression level of NPM-ALK.

AlphaScreen assays were performed as described previously [23 (link)]. All recombinant proteins used here was synthesized using a wheat germ based cell-free system as described above. For each protein kinase, 1 μl of crude recombinant biotinylated construct from the human kinase library was incubated with 1 μl of crude GST-Gag or GST-DHFR in 10 μl of kinase assay buffer (100 mM Tris–HCl pH8.0, 10 mM MgCl₂, 0.1% Tween20, 0.1% BSA) at 37°C for 1 h in one well of a 384-well Optiplate (Perkin Elmer, Foster City, CA. In accordance with the AlphaScreen IgG (protein A) detection kit (Perkin Elmer) instruction manual, 15 μl of detection mixture containing 100 mM Tris–HCl pH 8.0, 0.01% Tween-20, 1 mg/ml BSA, 5 μg/ml Anti-FLAG antibody (GE healthcare, Buckinghamshire, UK), 5 ng streptavidin-coated donor beads and 5 ng anti-IgG (protein A) acceptor beads were added to each well followed by incubation at 26°C for 1 h. AlphaScreen signals from the mixture were detected using an EnVision device (PerkinElmer) with the AlphaScreen signal detection program.