Ha peptide

NELF-E cDNA was purchased from Source BioScience LifeSciences (Clone IRAUp969C0381D) and cloned into pOZ-FH-N16 (link). NELF-E was purified from Dignam nuclear extracts30 (link) derived from HeLa S3 cells stably expressing Flag- and HA-tagged NELF-E (eNELF-E) by two-step affinity chromatography16 (link). Nuclear extracts were first incubated with anti-FLAG antibody-conjugated agarose beads (see list of antibodies in Supplementary Methods) and the bound polypeptides were eluted with FLAG peptide (Sigma) under native conditions. The FLAG affinity-purified material was further immunopurified by affinity chromatography using anti-HA antibody-conjugated agarose beads (see list of antibodies) and eluted under native conditions using HA peptide (Roche). Five percent of FLAG and HA immunoaffinity-purified eNELF-E or mock immunoprecipitations from 4 l of culture were resolved on SDS–PAGE and stained with the Silverquest-kit (Invitrogen). The remainder of the eluate was stained with Coomassie-R250. Individual Coomassie-R250 stained bands or for closely migrating bands, regions of the gel, were excised and subsequently analysed by tandem MS at the Harvard Medical School Taplin Biological Mass Spectrometry facility, Boston, MA, USA.

Yeast expressing HA-Ub were homogenized using a beat beater and then solubilized in solubilizing buffer containing in 50 mM Tris, 50 mM NaCl, 10% Glycerol, 1% Triton X-100, pH 7.4 for 30 min at 4°C. Detergent was diluted to 0.1% Triton and solubilized material was clarified by centrifugation at 20.000 g and 4°C for 10 min. Supernatant was loaded onto Monoclonal Anti-HA-Agarose (Sigma, A2095) for 2 h at 4°C. The agarose was washed 10 times with solubilizing buffer containing 0.1% Triton X-100. Bound proteins were eluted with 4mg/ml HA peptide (Roche, 11666975001) and subsequently analyzed by SDS gel and western blotting using HA specific antibody (Roche, clone 12CA5, 11583816001).

Anti-P-gp mouse mAb C219 and C494 were purchased from CalBiochem (La Jolla, CA). The CASP3 inhibitor (SC-3075) and CASP1 inhibitor (SC-3071) were purchased from Santa Cruz Biothechnology (Santa Cruz, CA). The Anti-HA affinity matrix and HA peptide were purchased from Roche Applied Science (Mannhein, Germany). Except where noted, all the chemicals for gold nanoparticles and ligand synthesis were purchased from Sigma-Aldrich.

Transfection of FreeStyle™ 293 cells was done according to the manufacturer's instruction. The culture supernatants were harvested at 72h post transfection and filtered through 0.45μm filter unit before purification. The supernatants were concentrated with 10k MWCO centrifugal filters (Millipore) and dialyzed with PBS before further purification. Small-scale protein purification was performed according to the manufacturer's protocol for anti-HA affinity matrix (Roche). Briefly, samples were incubated with anti-HA affinity matrix and following three washes, specifically-bound proteins were eluted by HA peptide (Roche) in elution buffer and then dialyzed with PBS three times. The purity of each protein was analyzed using coomassie blue staining, Silverquest staining (Invitrogen) and SEC analysis.