Tnt quick coupled transcription transcription system

For the phosphorylation assay, STAT5 (produced in reticulocyte lysate) was incubated for 15 min at 30 °C in the presence or absence of HSP27 (produced in reticulocyte lysate) with JAK2 recombinant kinase (50 ng, 14-640, Merck Millipore) in a 15 µl of assay kinase buffer (Hepes pH 7.4, 10 mM, NaCl 50 mM, MgCl₂ 5 mM, MnCl₂ 5 mM, DTT 1 mM, NaF 10 mM) in the presence or absence of ATP (250 µM, #9804, Cell Signalling Technology) and the reaction was stopped by adding 2× Laemmli buffer.
In vitro transcription and translation reactions were performed using TNT Quick Coupled Transcription/Transcription System (L1170, Promega) as recommended by the supplier. Next, 1 µg of the plasmid DNA template was transcribed and the protein was translated at 30°C for 90 min.
For the de-phosphorylation assay, a phosphorylation kinase assay JAK2/STAT5 was first done in the presence of ATP as described above and the reaction was stopped by adding 20 µM of Ruxolitinib (sc-396768A, Santa-Cruz) for 5 min. Then, HSP27 (produced in reticulocyte lysate) was added or not to the samples and incubated for 10 min at 30 °C. The recombinant SHP2 (590 ng, SRP0217, Sigma-Aldrich) was added or not to the samples for 12 min at 30 °C and the reaction was stopped by adding 2× Laemmli buffer.