Nickel beads

For expression of double-tagged (FLAG-His) neurexin substrate, E. coli strain BL21 (Invitrogen) was transformed using the gene of interest subcloned into the pet21b expression vector (Novagen). The expression of proteins was induced for 4 h at 37 °C in 1 litre of LB/ampicillin containing 1 mM isopropyl 1-thio-β-D-galactopyranoside. Cells were lysed with 10 mM Tris pH 7.0, 150 mM NaCl, 1%Triton X-100, and complete protease inhibitor cocktail (Roche) and passed three times through a high-pressure homogenizer (Emulsiflex-C5; Avestin, Inc., Mannheim, Germany) at a pressure greater than 1000 psi. The proteins recovered were incubated overnight with the M2 anti-FLAG affinity resin (Invitrogen) and the bound proteins were eluted in batch using the acidic solution (1%NP40, 100 mM glycine at pH2.7). The elution fractions were pulled together and bound overnight to the Nickel beads (Invitrogen). The proteins were then eluted using 200 uM imidazole solution. The purity of the eluted material was confirmed by SDS-PAGE using Coomassie blue staining (Applichem, MO, USA).

A His-tagged fragment of TCF/Pan containing both the HMG and C-Clamp domains was purified from E.coli strain BL21 following IPTG induction for 4 hours @ 37° using column purification on Nickel beads (Invitrogen) with Immidazole elution. LB growth media supplemented with 10 uM ZnCl. dsDNA probes were purchased from IDT and labeled probe was tagged with a 5′ 700 IR moiety on both strands. Competition assays were performed using the LI-COR Odyssey Infrared platform, and infrared intensity of the IR dye-labeled probe/protein complexes were calculated using Image Studio 2.0. The IC₅₀ values were calculated using Prism 6 for Mac OS X (Graphpad Software, La Jolla California), as were the saturation binding curves. Three independent experiments were used to perform a least-squares non-linear fit. Binding reactions were performed as described in [44] (link), briefly, with 50 ug/ml poly(dIdC). 0.05% NP40, 50 mM MgCl2 and 3.5% glycerol in binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT). Each reaction, containing 6 pmol recombinant protein and 0–2.4 pmol competitor dsDNA (dose indicated in figure 4A) was incubated for 5 min on ice, 25 minutes at RT before 20 fmol IR-dye labeled probe was added and reactions were incubated for an additional 30 minutes. A complete list of the probes used can be found in Table S1.

Cell lysates were prepared with cell lysis buffer (20 mM Tris-Cl pH7.4, 25 mM NaCl and 0.1% NP40) containing proteinase inhibitors. Proteins (2 mg) were incubated with control IgG, monoclonal anti-STIP1antibody (Abnova) or anti-HSP90 antibody (Abcam) at 4°C overnight under agitation. Enriched protein complexes were concentrated with protein A agarose beads (Millipore, Billerica, MA, USA). Agarose beads were washed for three times with wash buffer (20mM Tris-Cl pH7.4, 25mM NaCl) and boiled with sample buffer (0.25 M Tris-Cl pH 6.8, 0.08% SDS, 20% glycerol, and 10% β-mercaptoethanol). Immunoprecipitation was performed with an ImmunoCruz IP/WB optima system (Santa Cruz Biotechnology). Each sample was subjected to electrophoresis with 8% SDS-PAGE. Antibodies raised against JAK2, HSP90 (Cell Signaling Technology), STAT3 (Abcam), and STIP1 (Abnova) were used for western blot analysis. Halo fusion proteins, NTAP proteins, and His-tagged ubiquitin were pulled down using the Halo-tag protein purification system sample pack (Promega), streptavidin agarose (Sigma) and nickel beads (Invitrogen), respectively, following the manufacturer's protocols.