Tnt coupled transcription translation system

Equal amounts of GST and IbeA-GST fusion proteins were immobilized with Glutathione Sepharose 4B. Then different forms of Caspr1 protein derived from TNT coupled transcription/translation system (Promega), or from Caspr1 transfected 293T cells, or from HBMECs lysate, was applied and co-incubated with Glutathione Sepharose 4B prebound with GST-IbeA overnight at 4 °C, with GST as control. The bound proteins were washed with binding buffer (20 mM Tris, 50 mM NaCl, 10% glycerol, 1% Nonidet P-40) and the samples were analyzed by western blot. Each experiment was repeated at least three times.

Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors for 2 h on ice, with brief vortexing every 10 min. The lysates were centrifuged at 15,000 g for 20 min at 4°C to remove cell debris. Supernatants were incubated with the indicated antibodies or affinity beads at 4°C for 2 h, with gentle rotation. The immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting.
GST fusion proteins were expressed in Escherichia coli BL21 and purified using glutathione-Sepharose 4B (GE Healthcare) according to the manufacturer’s instructions. For the pulldown assays, glutathione beads were incubated with purified GST-tagged proteins in RIPA buffer containing 0.5% bovine serum albumin at 4°C overnight, with gentle rotation. In vitro-translated AR protein, which was produced by the TNT coupled transcription/translation system (Promega), was further incubated for 2 h. Bound proteins were analyzed by SDS-PAGE and immunoblotted with an anti-HA antibody.

Glutathione S-Transferase (GST) and GST-fusion proteins were purified from Escherichia coli BL21 (DE3) codon plus strain extracts, with glutathione-Sepharose 4B beads (GE Healthcare). Beads carrying the GST or the GST-fusion proteins were equilibrated in the following binding buffer (10 mM Tris-HCL pH 8, 250 mM NaCl, 0.1% NP40 and 2 mg/ml bovine serum albumin (BSA)) in the presence of protease inhibitors (complete EDTA-free cocktail from Roche Molecular Biochemicals) and incubated with radiolabeled proteins synthesized in vitro in the presence of [³⁵S]-methionine using the TnT Coupled Transcription/Translation system (Promega), in binding buffer for 4 h at 4°C. Beads were washed 5× in binding buffer without BSA and bound proteins were fractionated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and revealed by autoradiography.

Plasmid constructs encoding full-length Lhx2 protein, a truncated form that lacks the N-terminal Lim domain (Lhx2-ΔLim) or a homeodomain-deficient Lhx2 (Lhx2-ΔHD) have been described previously (Honda et al., 2012 (link)). The proteins were produced in vitro using the TNT coupled transcription-translation system (Promega). For the gel shift assay, double-stranded oligonucleotide probes containing the published Lhx2 binding site and the second putative site, in the 6.4 kb downstream region of Dmrt5 (5′-AGTTGCCTAATTCCACTTTAATTGGAAAGG-3′), or their mutated versions (5′-AGTTGCCGCCGGCCACTTGCCGGGGAAAGG-3′), were generated by annealing complementary oligonucleotides and labeling them with [γ-³²P] ATP and T4 polynucleotide kinase. Protein–DNA complexes were formed by incubation of 3 μl of in vitro translated protein with 50,000 cpm of the radiolabeled DNA probes for 20 min at RT in 20 μl of binding buffer (20 mm HEPES, pH 7.3, 60 mm KCl, 1 mm DTT, 1 mm MgCl₂, 0.1 mm EDTA, 10% glycerol, 0.5 mg/ml BSA) containing 1 μg of poly(deoxyinoinic-deoxycytidylic) acid sodium salt. The DNA–protein complex was resolved on a 6% native PAGE in Tris-glycine 1× buffer. The gel was fixed in 10% acetic acid and 10% methanol, and then dried. The complex formation was assessed by autoradiography. Electrophoretic mobility shift assay (EMSA) was performed in two biological replicates.

The murine NICD-4 cDNA corresponding to a truncated Notch4/Int3 cDNA (residues 4382–6043) has been described^{15 (link),17 (link)}. An oligonucleotide encoding hemagglutinin (HA) tag was added to the 3′ end of NICD-4/Int3 cDNA. The HA-tagged NICD-4/Int3, RAM 23, CDC10/ANK, RAM-ANK and PB-PEST expression vectors were generated through PCR using Pfu polymerase from StrataGene (La Jolla, CA, USA) and cloned into the eukaryotic expression vector pcDNA3 as described previously^{44 (link)}. The nucleotide sequence of all NICD-4/Int3 deletion plasmids have been determined and their sequences verified. The Int3 PCR deletion products were cloned into the NheI and XhoI site of the pcDNA3.1(−) (Invitrogen, Carlsbad, CA, USA). The protein encoded by the PCR product was synthesized using TNT Coupled Transcription/Translation System (Promega, Madison, WI, USA) according to the manufacturer’s specifications and as described previously^{45 (link)}. The synthesized protein was analyzed by electrophoresis on a 10% SDS-polyacrylamide gel (SDS-PAGE) and autoradiography. To measure the ability of the Int3 deletion mutants to activate NF-κB P50 signaling in HC11 cells containing an NF-κB reporter gene (Qiagen, Valencia, CA), cells were transiently transfected with expression vectors containing the Int3 deletions.