Tnt sp6 coupled reticulocyte lysate system

Recombinant GAD65 and IA-2 were labeled with ³⁵S-methionine (PerkinElmer, Waltham, MA, USA) by in vitro coupled transcription and translation using the TNT SP6 coupled reticulocyte lysate system (Promega, Madison, WI, USA) as described [14 (link)]. Full length cDNA coding for human GAD65 in the pTNT vector (Promega) (pThGAD65) or the intracellular domain (amino acids 606-979) of IA-2 in the pSP64 Poly(A) vector (Promega) (IA-2ic) were used as templates [15 (link)]. GADA and IA-2A were analyzed in a radioligand binding assay (RBA) [14 (link)]. Duplicate samples were incubated with radio-labeled antigen. The samples were transferred to filtration plates (Millipore, Solna, Sweden) and IgG antibodies precipitated with Protein A Sepharose (Zymed Laboratories Inc, San Francisco, CA, USA). After washing to remove all unbound antigen supermix scintillation cocktail (Perkin Elmer) was added and the radioactivity counted in a Wallac Microbeta Trilux system (Perkin Elmer). GADA and IA2A levels were expressed as units per mL (U/mL) derived from the WHO standard 97/550. GADA levels >34 U/mL and IA-2A levels >5 U/ml were considered positive.

³⁵S-labeled proteins (TBX5, NKX2-5, RBPjk, NICD, BAF60c serial deletions, Myocardin serial deletions) (Wang et al., 2001 (link)) were synthesized with the TnT SP6 coupled reticulocyte lysate system (Promega, L4600) or TnT T7 coupled reticulocyte lysate system (Promega L4610) and labeled with ³⁵S methionine (Perkin Elmer NEG709A). 5 µl of each synthesized protein was analyzed with SDS-PAGE gel and exposed to X-ray film for evaluation. GST-BAF60c, GST-RBPjk, GST-TBX5 and GST were expressed in E. coli strain BL21 and purified with glutathione Sepharose 4B (GE Healthcare, 17-0756-01). The beads were incubated with ³⁵S-labeled target proteins overnight at 4°C and washed with PBST for three times. The beads were then boiled in loading buffer. The protein was analyzed with SDS-PAGE gel and autoradiography.

In a 30-µl reaction, 2 μl of in vitro translated HA-TBP (generated using TNT SP6 Coupled Reticulocyte Lysate System (L4600, Promega, Madison, WI)) or 200 ng of recombinant GST-TBP was incubated with an ATP regenerating system (37.5 mM creatine phosphate, 5 mM ATP, pH 8.0, 5 mM MgCl₂). 2 μg of ubiquitin (U-100, Boston Biochem, Cambridge, MA), 50 ng E1 (E-305, Boston Biochem), 100 ng UbcH5b (E2-622, Boston Biochem), 2 μM ubiquitin aldehyde (U-201, Boston Biochem), 10 μM MG132 (474790, EMD Millipore) and 10 μg of S100, partial purified fractions or purified recombinant Huwe1 protein for one and a half hour. For detection of ubiquitinated species using the anti-ubiquitin antibody, after the reaction, reaction mixtures were diluted in buffer (1% NP40, 150 mM NaCl, 50 mM Tris–HCl pH 7.5, 0.5% sodium deoxycholate), followed by immunoprecipitation using anti-HA antibody/protein G sepharose beads or pull-down using glutathione sepharose beads. Purified TBP proteins were then subjected to three washes with the same buffer, eluted by boiling in 2×SDS loading buffer and analyzed by western blots. For detection of ubiquitinated species using the anti-TBP antibody, reactions were terminated with 2×SDS sample buffer, and then analyzed by western blots.

Oligonucleotides and their complements (Supplemental Table 1) were synthesized (Operon) with or without biotin end-labeling. The LSF binding sequence from the SV40 promoter was used as a positive control [24 (link)]. After mixing, each oligonucleotide pair was boiled at 100°C and cooled down to RT for annealing. Recombinant LSF protein was prepared using the TnT SP6 Coupled Reticulocyte Lysate System (Promega). EMSA was performed using the LightShift Chemiluminescent EMSA kit (Thermo Scientific) according to the manufacturer's instructions. DNA-protein complexes were separated by electrophoresis on 7% PAGE in 0.5 x TBE buffer, transferred to Hybond-n+ (GE Healthcare), and visualized using the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific).

The TnT® SP6 Coupled Reticulocyte Lysate System (Promega, cat#: L4600) was used to generate the protein products expressed from the QXQ readthrough plasmid (pDB603)^{41 (link)} which contains a UGA(C) PTC sequence. A 25 kDa protein was produced by efficient termination at the PTC, while a 35 kDa protein was generated by readthrough of the PTC. The Transcend™ tRNA (Promega, cat.# L5061) was added to the system for the chemiluminescent detection of the protein products via incorporation of biotinylated lysines. The assay was performed according to manufacturer’s instructions. The readthrough agents G418 or SRI-41315 were added to the reactions at 0.2 and 0.4 µg/ml, or at 5 and 10 µM, respectively. The reactions were incubated at 30 °C for 90 min and then 2 μL from each reaction was loaded onto precast 4–15% polyacrylamide gels (Bio-Rad, cat#4568084) to separate proteins via SDS-PAGE electrophoresis. The proteins were then transferred to Immobilon-P membrane-FL (Millipore), which was incubated with the Streptavidin-HRP conjugate (1:10,000 dilution) for 60 min, washed, and then incubated with SuperSignal^TM West Femto Maximum Sensitivity Chemiluminescent HRP substrate (ThermoFisher Scientific cat#34094) for 1 min. A Chemi-Doc^TM Imager (Bio-Rad) was used to detect the signal.

GST fusion proteins were in vitro transcribed/translated using TNT SP6-coupled Reticulocyte Lysate System (Promega) from pXen1 (for GST), or pXen plasmids encoding the indicated Msi protein. 5′ biotin-labeled RNA oligonucleotide probes were synthesized by Integrated DNA Technologies and corresponded to the last 50 nucleotides of the Xenopus Mos 3′ UTR (Accession NM_001088094), a mutant Mos 3′ UTR encoding a disruption of the consensus MBE^{33 (link)} or the last 90 nucleotides of the murine Pou1f1 3′ UTR which encodes a consensus MBE (Accession BC061213). Binding reactions, electrophoresis and probe detection were performed as described^{33 (link)}.